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From Whispering Gallery Mode Resonators to Biochemical Sensors

Médéric Loyez,* Maxwell Adolphson, Jie Liao, and Lan Yang

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ABSTRACT: Optical biosensors are frontrunners for the rapid and real-time
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detection of analytes, particularly for low concentrations. Among them,

whispering gallery mode (WGM) resonators have recently attracted a
growing focus due to their robust optomechanical features and high
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sensitivity, measuring down to single binding events in small volumes. In this

review, we provide a broad overview of WGM sensors along with critical
advice and additional “tips and tricks” to make them more accessible to both
biochemical and optical communities. Their structures, fabrication methods,
materials, and surface functionalization chemistries are discussed. We
propose this reflection under a pedagogical approach to describe and
explain these biochemical sensors with a particular focus on the most recent
achievements in the field. In addition to highlighting the advantages of WGM
sensors, we also discuss and suggest strategies to overcome their current
limitations, leaving room for further development as practical tools in various applications. We aim to provide new insights and
combine different knowledge and perspectives to advance the development of the next generation of WGM biosensors. With their
unique advantages and compatibility with different sensing modalities, these biosensors have the potential to become major game
changers for biomedical and environmental monitoring, among many other relevant target applications.
KEYWORDS: Whispering gallery modes, Optical resonators, Biosensing, Biochemical sensors, Microbubble, Microsphere, Microring,
Microtoroid, Microbottle, Racetrack resonator, Optofluidics

R ecent years have marked a global push toward developing

more effective biomolecular sensing technologies. The
COVID-19 pandemic, growing number of cancer cases, and
a cavity was born. Since then, this principle has been exploited
in various configurations for a range of applications, which
include building sensors through electromagnetic waves such
environmental issues such as pollution in air, water, and soil as light (from UV to infrared), radio waves, microwaves, and
have underscored the urgent need for advanced detection even X-rays.9,10
methods for early disease diagnosis, food and water safety Optical biosensing platforms are game-changing technolo-
monitoring, and detection of biochemical agents for defense gies that enable label-free molecular detection using versatile
purposes. While traditional gold-standard techniques remain at structures at micro- or even nanoscales. WGM resonators
the center stage (e.g., polymerase chain reaction, PCR; include many geometries such as microspheres, micro-
enzyme-linked immunosorbent assay, ELISA; paper-based capillaries, microbottles, microbubbles, microtoroids, and
rapid diagnostic tests, RDTs), emerging technologies are many other configurations. They support highly confined
starting to stand out. This is driven by the growing need for optical modes in small volumes with long photon lifetimes
sensors in various fields.1−4 Many optical techniques are thanks to the high-quality factor of the structures. The strong
currently in the pipeline with increasing interest, such as light and matter interaction makes them highly sensitive to the
surface Raman scattering (SERS), plasmonic optical fibers, and surrounding environment, inspiring a variety of sensing
enhanced fluorescent assays, among many others.5−8 One of applications, such as the detection of extremely small refractive
these many promising technologies is based on whispering index (RI) variations. These sensors are also sensitive to other
gallery mode (WGM) and is the topic of this review. parameters such as temperature, pressure, and mechanical
The history of whispering gallery sensors can be traced back
to the discovery of whispering-gallery waves in St. Paul’s Received: December 31, 2022
Cathedral in London (UK) by Lord Rayleigh (John William Accepted: June 9, 2023
Strutt) who found that whispers (acoustic waves) could be Published: June 30, 2023
heard across certain positions inside the dome. He then
developed wave theories at the beginning of the 20th century,
and the physics of resonance based on wave interference inside

© 2023 American Chemical Society https://doi.org/10.1021/acssensors.2c02876

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Figure 1. Working principle of a WGM sensor. (a) Schematic of a microbubble resonator and (b) of its cross-section (2D and 3D). The overlay
shows the z-component of the electric field representing the propagation of a WGM inside a microbubble resonator. The included inset is the
intensity map in 3D, showing the field maxima (red) and the field minima (in blue). This field is excited with an evanescent field coupling from a
tapered optical fiber. (c) Mode shift principle. (d) Mode splitting principle. (e) Mode broadening principle.

constraints and can therefore find their place in numerous wavelengths, light returns back upon itself in phase after
applications.11,12 It is now well established that these types of traveling for one round. As a result of constructive interference,
(bio)sensors are key enabling technologies to the development light satisfying the condition can be self-reinforced, while
of new sensing modalities, although not very widespread at the others are eliminated. This condition is called the optical
commercial level yet, with only a few companies and spin-offs resonance condition:
stepping into this market.13,14 WGMs have been extensively
studied for the detection of biomarkers at low concentration m = 2 Rneff
but also to spot single molecules, and are therefore a growing
hope to reach new sensing limits.15,16 Despite the many where m is an integer number, λ is the wavelength, R is the
aforementioned assets, they still suffer from delicate integration radius of the resonator, and neff is the effective refractive index.
and lack of reproducibility. New methods of interrogation and The self-reinforced modes are called whispering gallery modes
integration are needed to realize the potential for broad (WGMs), and the equation of the resonant condition provides
practical applications. All of these aspects will be thoroughly us with a simple and fundamental understanding of resonant
discussed in the following sections. modes in a WGM resonator. Any changes in the physical size
Because of the growing interest in WGM sensors, a great or refractive index, such as those induced by molecule bindings
deal of information has been disseminated in the literature so to the sensor or chemical changes in the local environments,
far. In this paper, we aim to combine and unify both will be manifested as a wavelength shift of the resonance, a line
engineering aspects and surface biochemistry in order to obtain width broadening, or a mode splitting of the resonance.
a common thread that can serve both scientific communities. Therefore, sensing with WGM-based sensors can be achieved
First, we describe the structures, methods, and materials used by monitoring the resonance shift. For instance, WGM
to manufacture WGM sensors. Then, we propose clear resonators have been demonstrated to measure the temper-
methods of surface biofunctionalization and discuss the recent ature in both laboratory and outdoor environments by tracking
state-of-the-art studies on this topic. We also explore new the changes in the resonant wavelength because the temper-
challenges of this fascinating field of research and provide ways ature dependence of the resonance arises from thermally
to investigate their integration. The use of microfluidics, induced changes in the refractive index and the physical
integrated 3D designs, and their benchmarking with laboratory dimensions of the resonator. This sensing method has been
standard bioassays are all topics that bring new insight and will used for various sensing applications. To describe WGMs in an
be covered throughout this review. accurate and rigorous way, wave optics theory can be used.

■ GENERALITIES ON WGM SENSORS

Working Principle (Theory/Explanation). Electromag-
Here, we apply wave optics theory for WGMs in a hollow core
microbubble resonator as an example (Figure 1a). As shown in
Figure 1b, the microbubble structure has an axial symmetry. At
netic waves can be described by wave equations similar to the cross-session view of a microbubble resonator, the
those for acoustic waves. In an optical WGM resonator, light structure consists of the core inside and the wall outside,
waves propagate near the periphery of the boundary of the surrounded by air. R1(z) and R2(z) are the inner and outer
resonator by a total internal reflection. If the effective length of radii, respectively. The refractive indices of the core, wall, and
the optical path in a round trip equals an integer number of air are n1, n2, and n3, respectively. The microbubble geometry
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Figure 2. Optical images of (a) microsphere, (b) microbottle, and (c) microbubble.

profile along the z direction, R2(z), can be described by the techniques will be provided below. Early research in WGM
truncated harmonic-oscillator profile: resonators primarily studied the emission characteristics of
liquid droplets.19,20 The advancement of fabrication techniques
R 2(z) = R b[1 + ( kz)2 ] 1/2
has enabled the fabrication of WGM resonators with various
where Rb is the radius at the equatorial plane (middle of the geometries, sizes, and materials. While many different geo-
microbubble) and Δk is the curvature parameter (a larger Δk metries can sustain the WGM, they typically have circular cross
means a larger curvature in the geometry). WGMs in such a sections that enable light to be trapped as it propagates near
structure with axial symmetry can be analyzed using the the periphery. To excite WGMs, coupling efficiency is crucial
Helmholtz equation (wave equation) in cylindrical coordi- and can be achieved by the prism technique or by optical fibers
nates: such as tapered fibers. Prism coupling is a key strategy that was
2 2
well described in early studies of surface plasmonic polariton
1 ij E yz 1 E E and WGM resonators.21,22 To excite WGMs, the resonator is
jjr zz + 2 2
+ + n2k 2E = 0
r rk r { r z2 positioned in close proximity to the base of the prism. A laser
beam is incident on the base of the prism and undergoes totally
where k satisfies internal reflection onto a photodetector for measurement. At a
k 2 = k 2 + kz2 specific incident angle, light can tunnel across the gap into the
WGMs of the resonator, causing a sharp drop in the intensity
kz and kφ are the axial and azimuthal wave vectors. of the reflected light.23,24 The propagation constant of the
The radial distribution of the WGM electrical field of a evanescent field from the prism can be controlled by that
microbubble resonator is governed by incident angle, allowing for easy single-mode coupling through
l phase-matching. A variety of prism materials are available that
o
o AJm (k n1r ) r R1(z)
o
o are suitable for different wavelengths and resonator materials.
o
o
o
o (1) The major disadvantage of this method is that components are
Ez(R ) = m
o BJm (k n 2r ) + CHm (k n 2r ) R1(z) < r R 2(z)
o
o bulky and difficult to finely align with WGM resonators. Angle-
o
o
o
o DHm(1)(k n3r )
o
n
r > R 2(z) polished fiber (fiber prism) coupling uses a mechanism similar
to that of prism couplers, but with a much smaller footprint.
where Jm and Hm(1) are the mth Bessel function and the mth The end of an optical fiber is polished at a certain angle such
Hankel function of the first kind, respectively (m denotes the that the optical mode propagating in the fiber experiences a
order of the Bessel/Hankel function). The resonant wave- total internal reflection at the end of the fiber. When the
length can be obtained numerically from the equation by resonator is placed in proximity, the evanescent field at the end
matching the boundary conditions at R1 and R2.17,18 surface couples with WGMs at certain angles (phase-matching
2 neff R b condition). The angle-polished fiber is essentially a prism
mpq = coupler integrated with an optical fiber as the end of an optical
2
m + m(2q + 1) kR b
fiber is polished at a specific angle to enable coupling with
WGMs can be described by three modal numbers WGMs when the resonator is brought close, and the other part
(azimuthal m, radial p, and axial q), where 2m (m = 0, 1, 2, of the fiber plays the role of a waveguide. The method offers
···) describes the number of maxima along the circumference, p the advantage of easier alignment but requires precise control
(p = 1, 2, ···) describes the number of maxima along the radius, of the angle and fine polishing of the surface. Using the prism-
and q (q = 0, 1, 2,···) describes the number of maxima along coupling technique could therefore be helpful to solve issues
the axial direction (longitudinal direction). The WGM has two related to microfluidics implementation and sample delivery
polarization states: transverse electric (TE) modes and for certain WGM configurations for which an external
transverse magnetic (TM) mode. Using this three-layer packaging is needed to protect their integrity, such as
model, the WGM spectral position can be obtained as a microspheres, microbubbles, etc.25−27
function of wall thickness, resonator size, operating wave- Fiber-Based Resonators. Microspheres and microbottle
length, etc., which allows one to analyze the sensitivity to resonators are typically made from optical fibers (Figure 2a,b).
external changes and optimize the sensor design. Microspheres represent the simplest resonator geometry, and
In general, there are three types of mechanisms for WGMs their mode characteristics can be analyzed with rigorous wave
sensing, namely, mode shifting, splitting, and broadening optics theory or simulations based on Mie’s theory. Their
(Figure 1c,d,e). Further discussion about these demodulation fabrication is relatively easy by heating the tip of an optical
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Figure 3. SEM images of (a) microring, (b) racetrack, (c) microtoroid, and (d) microdisk resonators.

fiber via arc discharge, CO2 laser radiation, or hydrogen flame, bubble resonators provide additional confinement along the
and the tip of the fiber melts into a sphere under the action of capillary axis (Figure 2c). By heating and softening the
surface tension. By changing the heating temperature and capillary with a CO2 laser or filament while applying internal
heating time, microspheres with different diameters can be pressure, the capillary inflates and forms a bubble shape.
fabricated. Other materials such as polymers or gain materials Similar to microtoroid resonators, microbubbles show smaller
(such as erbium, Er3+, or ytterbium, Yb3+) can also be coated surface roughness after reflowing and exhibit higher Q-factors
on top of their surfaces, modifying the physical properties of (∼108) compared to those of the other structures. The
the resonator. Microbottle resonators have a cylindrical bottle capillary wall thickness can eventually be further thinned using
structure with a solid core. They can be fabricated by either hydrofluoric acid (HF) injected within its structure. This step
“heat-and-pull” techniques or “soften-and-compress” techni- is usually performed by withdrawing a solution at a constant
ques.28−30 Both techniques rely on softening of a small region flow rate using a microperistaltic pump or syringe pusher.34−38
of the optical fiber. The former sequentially tapers the fiber in On-Chip Resonators. On-chip WGM resonators are
two adjacent places to form the bottleneck, and the latter typically more stable and have proven the advantages of
compresses the fiber and forms the bottle. As for microspheres, mass production. Their on-chip integration benefits from
microbottles can be coated with other materials to tune their modern photolithographic technologies. They can be fab-
properties. It is worth noting that in bottle shaped resonators ricated in different material systems and form large sensors
(both microbottle and microbubbles, which will be further arrays. Planar microring resonators are the most widely used
described), the WGM spectrum typically appears very dense. structures in on-chip platforms (Figure 3a). They are curved
Such a rich variety of modes in the spectrum is useful in cavity planar waveguides and can be easily made in arrays. With
quantum electrodynamics (cavity QED) studies.30−32 sensor arrays, individual sensors can be used as an internal
Optofluidic Resonators. Optofluidic WGM resonators reference and eliminate common mode noise, while cascaded
have hollow core structures that provide them excellent microring resonators, which exploit the Vernier effect, can be
compatibility with microfluidic systems. They can be filled with used for sensitivity enhancement.
liquids or gases for sensing purposes without affecting the Racetrack resonators are another structure widely used in
external coupling. By contrast, other WGM sensors suffer from integrated photonics (Figure 3a,b). The straight region of the
flow fluctuations and potential contaminations when the racetrack is a typical planar waveguide, which provides a longer
coupling region is exposed to an aqueous environment. The path for light to travel and enhance the light−matter
integration of photonics and microfluidics into a single interaction due to the longer interaction length. The straight
component makes microcapillary and microbubble resonators region also makes it easier to engineer the properties of the
promising candidates for the development of optofluidic resonator. The modes in racetracks and microring resonators
sensors.33 Microcapillary resonators, which are also called are not “classic” WGMs in terms of total internal reflection
liquid-core optical ring resonators (LCORR), are typically from a single boundary. Here, the inner boundary reflections
made from silica capillaries. For enhanced performance, such are also essential for the formation of the modes.39 Compared
as higher Q-factors and sensitivities, LCORRs are often made with other on-chip WGM resonators, microring and racetrack
by pulling the capillary while softening the glass with a CO2 resonators have smaller mode volumes and lower modal
laser or a hydrogen flame. The hollow-core microcapillary density due to their waveguide nature which suppresses high
devices can be easily integrated with microfluidics. To confine order modes. Microdisk and microtoroid resonators are
WGMs in 3D rather than 2D for higher Q-factors, micro- fabricated from layers of silica on crystalline silicon substrates
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Figure 4. Schematic procedure for the photolithography process (on-chip resonators).

(Figure 3c,d). Silica disks are made by etching with geometry for a ring resonator or a simple line for a bus
hydrofluoric acid and subsequently undercut to form silica waveguide). Subsequent etching steps will remove the
disks on silicon. Compared with fiber-based WGM resonators, unwanted material(s), such as using a reactive ion etcher.
the fabrication of planar microring, racetrack, and microdisk The material that is not covered with a photoresist is removed
resonators is more controllable. However, the degraded surface to form the desired patterns. Lastly, with the photoresist
roughness resulting from the fabrication process limits their Q residual removed, an additional cladding layer is deposited on
factors to ∼106.40−42 the fabricated devices to ensure their protection.44
As an evolution of microdisks, microtoroid resonators Other Geometries. For most WGM resonators, the
combine the advantages of microring and microsphere
rotational symmetry and low radiation loss impede energy
resonators. Specifically, they possess a high Q-factor (∼108)
and exhibit simpler spectral characteristics compared to those exchanges between resonators and their surrounding environ-
of microsphere resonators.43−45 While the microdisks made of ment, resulting in a challenge when light is coupled from free
silica are heated with a CO2 laser, they are transformed into space into and out of resonators. Deformed sensors offer
toroids via a self-terminating process in which the CO2 laser feasible solutions as the excitation of WGMs and the detection
power is fixed. The transformation of toroids stops at the point of transmitted light can be accomplished via free space optics
where the heat loss from the silica exceeds the absorbed heat with high efficiency.49,50 These resonators are intentionally
from the laser. Microtoroid resonators have a smaller mode deformed to break their circular symmetry and enable free-
volume than microsphere resonators due to the small radius of space coupling using carefully aligned focused light. However,
the toroid transformed from the flat disk. Microdisks and the bulky size of the setup and alignment issues usually limit
microtoroids could potentially both be integrated and form their applications.
resonator arrays such as microrings and racetracks. However, Aside from “deformed resonators”, the Taiji resonators,
they are supported above the substrate by small pedestals sometimes referred to as “theta” or “yin-yang” resonators, are
inducing geometrical restrictions on the type of waveguides resonators with an S-shaped additional crossover branch
(suspended silica waveguides) used for WGM excitation.46 embedded in a microring. Such a resonator coupled to a
Usually, a fiber-taper waveguide is used to couple light in and
waveguide can be regarded as a non-Hermitian system. In the
out of the undercut microtoroid or microdisk resonators.47,48
To manufacture these on-chip structures, photolithography forward direction, light cannot propagate in the S-shaped
is one of the most commonly used techniques. It is indeed a waveguide, and the clockwise (CW) mode is not excited; in
strategy that easily lends itself to mass production in the field the reverse direction, light couples to the S-shaped waveguide,
of microfabrication. Gaining popularity in the field of and the counterclockwise (CCW) mode can be excited. In this
microelectronics and integrated circuits, it has also found way, nonreciprocal losses are introduced, and properties of
widespread applications in the field of integrated photonics. non-Hermitian physics can be found in the system. Taiji
The term photolithography refers to the selective transfer of a resonators show potential in unidirectional lasers, topological
pattern or mask to a chemical layer known as photoresist. The lasers, and unidirectional reflectors.51−54
photoresist can be divided into two categories, positive and Recently, microdroplet resonators have garnered attention
negative photoresist. Positive photoresist is broken down by due to their unique structures and ability to directly investigate
light exposure and is easily removed with a photoresist physical phenomena in liquid environments. However, these
developer. In contrast, a negative photoresist becomes structures are prone to evaporation and are less stable
insoluble in the photoresist developer when exposed to light. compared to solid-based geometries.55 They also rely on
The general process is as follows, but there are many variations lasing and require a gain of materials, consequently leading to
to this procedure depending on the specific application (Figure
more complex structures. Last but not least, they present more
4). Taking the fabrication of silicon photonic devices as an
complex fluidic handling, as they are themselves liquid.
example, the process typically starts on a silicon-on-insulator
(SOI) substrate, in which a thin device layer of Si (e.g., 220 nm Although they are less stable over time compared to solid-
thickness) is bonded on the top of a silica layer (several state resonators,56,57 they are interesting platforms to study
micrometers) on the Si substrate. The silica layer acts as an physical phenomena and biosensing in laboratory settings. The
insulator to protect the light in the device layer from leaking great advances and great promises of these sensors are their
into the substrate. Now lithography is used to transfer the ability to produce microstructures in vitro within cells and
desired pattern to the resist by UV exposure (mask aligner) or therefore analyze molecular events happening intracellularly
electron beam exposure (e-beam writer) (e.g., a circular through WGM excitation.58,59
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Figure 5. Fabrication, materials, and tips and tricks for WGM resonators.

■ FABRICATION, MATERIALS, AND COATINGS

Above, we described some commonly used materials such as
and is hydrophobic. But due to the polymer’s hydrophobic
nature, weak polar molecules such as methanol and ethanol
and nonpolar molecules such as ethylene can pass through
silica and silicon, along with their corresponding fabrication
PDMS. The interaction of weakly polar molecules and PDMS
techniques for WGM resonators. However, it is worth noting
causes swelling of the polymer and a refractive index change.
that other materials have been employed for WGM resonators,
When integrated with a microbottle resonator, both physical
for particular functionalization or better physical properties. In swelling of the polymer and refractive index change will cause a
this context, we introduce several common polymer materials shift in the resonant wavelength, enabling sensing. In Zhu et
and their corresponding fabrication techniques. al.,61 a single mode fiber (SMF-28) was dip coated in PDMS
Dip/Pull-Coating. The key step in manufacturing micro- and then baked to form a fully cured microbottle resonator.
resonators is reaching high quality and precision (Figure 5). The final diameter of the resonator could be controlled by
To reach such features, several fabrication techniques can be varying the standing time at room temperature of the uncured
exploited. Among them, the dip (pull) coating method is a PDMS resonators before curing. In the range of 4.19 to 41.90
simple and well-known method for the coating or fabrication ppm, the sensitivity of the Microbottle was 36.24 pm/ppm,
of optical resonators with polymers and other materials in and when the concentration was increased from 62.84 to
solution which can be subsequently cured (e.g., polydime- 272.35 ppm, the sensitivity decreased to 3.54 pm/ppm (see
thylsiloxane, glycerol, and sol−gel film). For all polymer more about sensitivity in Performance Indicators section).
resonators the surface tension of the liquid combined with the Sensing of nonpolar or weakly polar gas molecules can also be
force of gravity can create a near perfect spherical droplet. This performed in a silica microbottle coated with PDMS instead of
method requires the control of numerous parameters such as fabricating the resonator entirely out of PDMS. Wang et al.
dipping speed, rest time in the solution, pulling speed out of showed an SMF fiber locally heated by the arc discharge from a
the substrate, and rest time of the solution after dipping. All of fusion splicer to create a microbottle. It was functionalized
as these parameters can affect the physical properties of the with PDMS by pull coating. For concentrations of ethanol
resonator such as its final diameter. External parameters such between 5.8 and 117 ppm, the sensitivity of the PDMS-coated
as temperature, air flow and pressure, and abundance of silica microbottle was around 13.13 pm/ppm. For ethanol
particulate matter in air can also affect the final quality of the concentrations between 117 and 204.25 ppm, the sensitivity
coating.60,61 decreased to 1.44 pm/ppm. This research shows that a full
Polydimethylsiloxane (PDMS) is an elastic polymer that PDMS microbottle is more sensitive for weakly polar gas
shows high light transmittance and a strong thermal response sensing than a PDMS coating added on a silica microbottle.62
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Another possibility is to generate a microdroplet-based areas such as biomedical sensing and environmental monitor-
resonator. For instance, this can be achieved using glycerol, as ing.16,68,69
it has reached the highest sensitivity to relative humidity (RH) Thin Film Deposition. The fabrication of on-chip
(compared to other WGM resonators) thus far. It has reached structures that call on photolithography usually requires the
a sensitivity of 2.85 nm/% RH with a resolution of 1 × 10−4 % deposition of photoresist such as SU-8 (named from the
RH. Glycerol is especially suited for RH sensing as it contains presence of 8 epoxy groups). SU-8 is a polymer that can be
3-OH groups and is a trivalent alcohol, which means it can cross-linked with a photoinitiator such as UV light and has
easily absorb moisture from the air. A SMF fiber was tapered been used in the fabrication of polymeric WGM resonators.
and dipped into glycerol solution to form a nearly perfect The SU-8 microdisk, supported by a silicon pillar, is evaluated
droplet on the fiber tip due to the surface tension forces of on its sensitivity to selected compounds in their vapor phase
glycerol. The quality factor of the resonator was measured to (namely, humidity, isopropanol, toluene, limonene, 1-butanol,
be 7.3 × 103 when measured with a tunable laser centered and pentanoic acid). Out of these compounds, the SU-8
around 760 nm, and it is worth noting that this quality factor microdisk showed the greatest sensitivity (23 pm/ppm) to
increases for RH > 70%. Glycerol was a prime candidate for pentanoic acid, with a limit of detection of 0.6 ppm. It has also
RH sensing as it is a hygroscopic material, shows little been shown for polymeric resonators that the degree of
sensitivity to temperature, is transparent at 760 nm and shows attachment of the SU-8 disk to the supporting substrate can
selectivity to humidity.63 affect their overall sensitivity. For example, if a detection
In order to achieve high sensitivity, 2D-materials have scheme relies on physical deformation of the resonating
attracted considerable attention from the whispering gallery polymeric cavity, then maximizing this degree of freedom is
mode community.64 Graphene, being one of the most crucial. Researchers found that the deformation of a sensor can
intensively researched 2D-materials, has many exciting be improved by reducing the area where the SU-8 disk and
characteristics such as high sensitivity, arising from graphene silicon pillar meet since a large supporting pillar will reduce the
plasmons that range from terahertz to mid-infrared wave- overall movement of the disk. These microdisks were
lengths. Similar to graphene, graphene oxide (GO) is a single fabricated by spin coating SU-8 onto a silicon wafer to form
layer of carbon atoms covalently tethered by oxygen an ∼700-nm-thick layer. The polymer layer was selectively
derivatives.65 GO has been an interesting candidate for surface cured into disks with UV light and photoresist. Lastly, the
functionalization of gas sensors due to its large contact area silicon substrate was etched away with SF6 by reactive-ion
with gas molecules. Its functional groups and the fact that GO etching to form the supporting pillars.70
changes its conductivity with the adsorption of gases are other Spin coating is a commonly used method when the precise
key assets, consequently leading to refractivity change (as control of thin film coating thickness, such as SU-8 or sol−gel
conductivity is related to the imaginary part of the film, is critical.69,71 In spin coating, the material is deposited on
permittivity).66,67 In 2021, Wang et al. functionalized the the center of a wafer (commonly silicon) which is then spun
outer surface of a silica microbottle WGM resonator for up to 10,000 rpm. The film thereby experiences the centrifugal
ethanol sensing. The researchers achieved a sensitivity of 1.55 force and spreads outward until the desired thickness is
pm/ppm for ethanol concentrations ranging from 30 to 210 achieved; faster rotation results in a thinner final layer on the
ppm.62 substrate. If the film to be deposited consists of a nonvolatile
In sensing applications, the ability to detect small changes in solute and a volatile solvent, the thinning will also be a
the resonance is crucial for achieving a high performance. By function of evaporation as well as hydrodynamic force caused
increasing the Q-factor of a resonator, it becomes possible to by the rotating substrate. Spin coating of thin films is used in
resolve smaller changes in the transmission spectrum, which the fabrication of “on-chip” resonators such as microtoroids
leads to improved sensitivity and accuracy in sensing. By and microdisks, but it does not define the fabrication process
introducing a gain medium into the resonator, the losses can of these geometries. On-chip resonators typically utilize the
be compensated by the gain, which can increase the Q-factor spin coating process for thin film deposition, but the formation
and narrow the line width. This technique is commonly used in of subsequent geometries is achieved through an etching
laser resonators, where a gain medium such as erbium-doped step.72
solgel films is coated on the surface of WGM resonators, such Atomic layer deposition (ALD) is another method used for
as microspheres and microtoroids. Laser dynamics can be the formation of thin films on a substrate. Unlike other
adjusted by varying the doping concentration and thickness of chemical vapor deposition (CVD) methods that inject all
the solgel layer for continuous-wave or pulsating modes. This precursors at the same time and do not rely on the specific
technique allows for single-line laser emission and a low- order of the precursors and their reactions, ALD relies on the
threshold operation. Due to the improvement of Q-factors via interaction of the precursors with the substrate in a specific
gain medium, the WGM microlaser can detect individual sequence. In this way, the precursors and substrate react with
particles as small as 15 nm radius polystyrene nanoparticles, 10 each other specifically to form a deposited layer. Self-limited
nm gold nanoparticles, influenza A virions in air, as well as 30 growth of monolayers is controlled by self-saturating chemical
nm polystyrene nanoparticles in water with high sensitivity. reactions of the precursors with the available surface groups on
The detection method relies on measuring changes in the beat the substrate. Using ALD, metal−organic frameworks (MOFs)
note produced when an ultranarrow emission line from the can be deposited. They make up a class of compounds that
microlaser is split into two modes by a nanoscale object, which consist of metal ions held together by organic ligands and have
then interfere with each other. The laser line width, which can been used for gas selectivity in rolled-up microtube WGM
be much narrower than the resonance line width of any passive resonators. While gas sensing with unfunctionalized WGM
resonator, sets the ultimate detection limit. This highly resonators has been difficult in the past due to the sparse
sensitive detection capability of WGM microlaser-based nature of gaseous media, researchers have been utilizing MOFs
detection could have significant potential for applications in for resonator functionalization to imbue selectivity. Zeolitic
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Figure 6. (a) Typical spectrum obtained from a silica-based microbubble resonator coupled with a taper fiber. Adapted from Murugan et al.,76
Copyright 2011 Optical Society of America. (b) Transmission spectrum of a high Q-factor microsphere resonator. Reproduced with permission
from Dong et al.,77 Copyright 2009 Chinese Optics Letters. Typical spectra obtained from a (c) microring and (d) microtoroid resonators, adapted
from Luo et al.78 Copyright 2011 Optical Society of America and from Armani et al.79 Copyright 2005 AIP Publishing. Pictures from optical
microscope (a and b) and scanning electron microscope (SEM) (c) and a 3D drawing of microtoroid (d).

imidazolate framework-8 (ZIF-8), which is a subclass of a transparency window. This is difficult to achieve in a single-
MOFs, can effectively adsorb CO2 molecules since the pore waveguide-coupled microcavity system because any optical
size of ZIF-8 is comparable to that of a CO2 molecule (3.4 Å). perturbation will shift the modes in the same direction. By
The researchers note that although other functionalization adding a thin layer of PDMS to a silica toroid, WGMs can
schemes have been shown before for the purposes of CO2 propagate in both the PDMS and silica simultaneously such
sensing, the diversity of MOFs and related materials might be that two modes in the resonator can be thermally tuned to
capable of detecting other gases, as well. The functionalized overlap because of the large negative thermo-optic coefficient
rolled-up microcavities were made of Y2O3/ZrO2 which were of PDMS and the small positive thermo-optic coefficient of
subsequently sandwiched between two films of ZIF-8 by ALD. silica.75
Recent works showed the highest optical sensitivity to CO2
achieved so far, reaching 188 nm per refractive index unit
(RIU) with a limit of detection (LOD) of 5.85 × 10 −5 RIU.73
■ SENSING MECHANISMS AND SPECTRAL
ANALYSES
Surface Wetting. The coating/film formation methods Typical WGM Resonances. Typical whispering gallery
mentioned above rely on the substrate (material to be coated) mode spectra include numerous narrow resonances, as
being homogeneous to ensure high-quality coating. However, depicted in Figure 6, obtained from several types of WGM
not all WGM resonators have smooth homogeneous surfaces, resonators, from a microbubble (Figure 6a), a microsphere
such as the microtoroid geometry. Due to the signature bulged (Figure 6b), a microring (Figure 6c), and a microtoroid
toroidal edge, classic thin film deposition methods can lead to (Figure 6d). Typically, the entire spectrum within one free
inconsistencies in the coating near the edges and overhang of spectrum range or across the tuning range of the laser source
the microtoroid. To address these issues, other methods of would be collected, and the “best” mode would be identified in
surface coating, such as surface wetting, have been developed. post analysis by observing which one shows the largest
Surface wetting relies on the coating material having a low response to the measurand of interest, whether that response is
surface tension such that it will completely wet a silica a mode shift, mode split, or mode broadening. This
resonator when brought into contact with the toroid edge. Due identification method, to reach the most sensitive WGM in a
to the small scale of a microtoroid geometry, precise handling sensing experiment, can be universally applied to any
and alignment of the droplet in relation to the microresonator geometry. Once the most sensitive WGM or WGMs have
are crucial. been identified in the collected spectra, the following analysis is
Surface wetting can be used to create a smoother resonator straightforward. For instance, a mode shift can be tracked over
interface and modify its properties. PDMS coatings on silica time in the case of a bulk refractive index change. This can be
resonators have been shown to exhibit electromagnetically done manually or with custom or commercially available peak
induced transparency (EIT).74 In order to achieve an EIT, two tracking softwares. Mode splitting or broadening can also be
modes must be tuned to overlap such that there is destructive tracked over time and ultimately related back to the shift of
interference between the two modes in the resonator, creating interest. The overall spectral analysis is more straightforward
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Figure 7. (a) Optical WGM barcode sensing system. Schematic of WGM sensing using the optical barcode technique. The light is coupled into and
out of the microbubble via a tapered fiber. (b) Optical micrographs of the capillary (left) and fabricated microbubble (right). (c) Schematic of the
packaged substrate containing the microbubble and tapered fiber. (d) Schematic of the setup used. A tunable laser is used to probe the spectrum of
the packaged WGM sensor. The light intensity is controlled by the attenuator, and the transmission spectrum is received by a photodetector, which
is connected to an oscilloscope and a computer. Reproduced with permission from Liao et al.83 licensed under a Creative Commons Attribution 4.0
International License.

Figure 8. (a) Series of normalized transmission spectra presenting mode splitting, at 1550 nm with corresponding optical images for successive
depositions of KCl nanoparticles (from top to bottom, without nanoparticles to 4 nanoparticles). Reproduced with permission from Zhu et al.87
(b) Series of transmission spectra presenting mode broadening, at 1550 nm for successive deposition of nanoparticles (from top to bottom, without
to 4 nanoparticles). Reproduced with permission from Shao et al.88 Copyright 2013 WILEY-VCH Verlag GmbH and Co.

for microdisk and microtoroid resonators, which typically have standardized conditions to avoid any fluctuations from external
a sparse modal density compared to microbubble/microbottle parameters. Reference levels against drifts due to laser
or microsphere counterparts. However, despite their more instability or temperature changes can be essential to achieve
complex spectra, these dense modal systems offer rich high-precision biochemical sensing and self-reference of the
information that can be exploited through advanced signal system. WGMs with a high-quality factor are also better suited
processing techniques. Therefore, careful analysis of the dense for molecular detection as it is easier to resolve smaller shifts
spectra of microbubbles, microbottles, and microspheres may when the line width is narrower. A few common sensing
yield additional insights that are not available in the sparser applications that use mode shifting for detection include
spectra of microdisks and microtoroids. displacement,80 temperature,81 and biomolecule binding,82
Mode Shifting. Tracking the shift in wavelength of a among others.
resonant mode is one of the most common methods used in Recent advances have bolstered the idea of tracking a WGM
WGM sensing due to its ease of implementation and the broad further to enable what is called “barcode measurements”. As
range of both physical and biochemical analytes it can detect. mentioned above, tracking a single WGM mode offers only a
However, perturbations to the local optical environment such relative quantification of the target of interest, without a known
as changes in the bulk refractive index or temperature can baseline against which to reference the mode shift against.
modify the resonance condition, leading to a shift in the Second, the dynamic range of a measurement is limited by the
resonant mode. It is therefore important to work under scanning range of the laser. So, once the WGM shifts out of
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frame, the measurement is over. It is however possible to measuring wavelength shift.89 These single molecule detections
increase the scanning range of some laser sources, but this will are further described in this review.
be accompanied by a corresponding decrease in resolution. Performance Indicators. Each biosensor is characterized
The WGM barcode method addresses both of these issues. In and appreciated by its performance, more specifically through
this method, instead of tracking the shift of a single resonant its sensitivity and LOD. However, this is often a shortcut
mode, the changes in the entire spectrum are recorded to because many other indicators exist and all heavily depend on
enable sensing. Before measurement of an unknown target is the experimental conditions. The medium used for its
performed, multiple spectra are recorded over a predefined characterization (water, blood, food sample, etc.) and the
temperature range. Each spectrum collected is then turned into biotarget itself (protein, DNA, cells, etc.) can both influence
a “barcode” for that particular temperature and will collectively those parameters and should therefore be considered. The
form a catalog for future unknown temperature measurements, main performances indicators are sensitivity, LOD and limit of
such that, when a WGM spectrum is recorded for an unknown quantification (LOQ), specificity, accuracy, repeatability/
temperature, the barcode generated can be referenced against reproducibility, figure of merit (FOM), and quality factor
the catalog to enable absolute temperature sensing (Figure 7). (Q-factor). All these parameters are mainly dependent on the
Lastly, since this method does not rely on the tracking of a bioavailability of the target inside the medium but also on
single mode, the dynamic range of the measurement can be temperature, volume, and efficiency of the biofunctionalization
extended.83 process, among many others. First, the sensitivity can be
Mode Splitting and Mode Broadening. WGM operate defined as the reported shift of a monitored parameter (usually
under a degeneracy where two counterpropagating modes a wavelength shift for WGM resonators) depending on the
(CW and CCW) share a single eigenfrequency. This concentration of analytes or the refractive index change (RI)
degeneracy can be broken by the introduction of a inside a specific medium. Optical biochemical sensors often
subwavelength scatterer which couples the counterpropagating need both bulk and surface characterization, depending on the
modes, leading to mode splitting (Figure 8a). Not only can external matrix used.90,91
mode splitting detect a microscatterer but it can also estimate
resonance resonance
the size of the scatterer based on the amount of mode splitting Sensitivity = =
that is measured. Further, unlike mode shifting, mode splitting RI analytes
is immune to external changes of the environment, such as
temperature and pressure, and from noise generated by the The limit of detection is expressed in units of concentration
laser source, as it is a self-referenced measurement. As an and is defined as “the smallest analyte concentration that a
example, mode splitting has been used for the detection and given analytical system is able to distinguish with reasonable
size quantification of polystyrene (PS) particles in air and reliability from a sample without analyte”. The signal-to-noise
water.84,85 ratio is, therefore, an important parameter to optimize. Despite
Similar to mode splitting, measuring the change in line width this, the LOD often leads to unrealistically low estimations
of a WGM is also a self-referenced measurement and is most because it is often presented indirectly through a linear
commonly used to measure scattering or absorption losses regression performed on a set of measurements, assuming that
from nanoparticles or biomolecules. Mode broadening (Figure the sensitivity is quasi-constant and the sensor is under optimal
8b) is impervious to environmental perturbations and noise conditions. However, it may also take into account the
from the laser source itself, and it removes the requirement of dynamic range of the interactions and be experimentally
ultrahigh-quality factors that are beneficial when measuring verified.81,92−94 The most common method to determine the
mode shifting and splitting. Despite the commonly used term theoretical LOD is to report 3× the standard deviation in blank
“mode broadening” the line width of the mode can grow or buffer on a detection curve, leading to an intersection giving an
shrink in response to surface binding depending on the approximated concentration as the smallest detectable by the
location of the binding event.86 sensor. To be fairly estimated, the sensing curves need
While both mode splitting and mode broadening can be sufficient data points to fit a reliable linear or sigmoidal curve.
used for the detection of nanoparticles, Subramanian et al. It is also possible to summarize the LOD as 3 times the
standard deviation divided by the sensitivity. Another
recently showed a combination of these two methods for single
parameter tightly linked to the LOD is the limit of
molecule (sub kDa) detection. Here, gold nanorods are
quantification (LOQ). It is defined as the lowest level at
randomly attached to the surface of a glass microsphere for the
which an analyte can be quantitated with any degree of
plasmonic enhancement of the local electric field. The idea is
certainty. The determination of the LOQ is similar to that of
that molecules can be detected by interaction with the WGM
the LOD so they are often reported together. One common
resonator. In the past, single molecule detection experiments
method consists of reporting the signal-to-noise ratio of 10:1
have solely relied on mode shifting measurements, because
or to find an approximation by multiplying the LOD by 3. By
small molecules and proteins show weak polarizability. But
extension, the LLOQ (lowest calibration standard on the
rather than solely relying on mode shift for single molecule
calibration curve) can be mentioned. The latter is reported as
detection, the researchers also measure an effective change in
the detection response at least five times over the blank
the WGM line width. This effective change in line width is the
measurement.95
result of a frequency splitting of the standing wave modes
caused by the binding events to the gold nanorods. However, LOD = in blank + 3·(max sd)
because the splitting is smaller than the line width of the
modes, what is observed is an effective broadening or or
narrowing of the line width. By tracking the effective change 3·(sd)
in the line width of the WGMs, the researchers showed a larger LOD =
signal amplitude from the binding compared to that of Sensitivity

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LOQ 3·LOD ment, etc.), reproducibility refers to results obtained at

different locations, with different instruments, and conducted
The figure of merit (FOM) for a wavelength shift is equal to
by different individuals. It therefore measures the ability to
the ratio between the sensitivity (nm) and the line width of the
replicate research from one laboratory to another.98
resonance, considering that it is easier to measure the exact
After these considerations, it is important to mention that
location of a narrow resonance than a broad one.
the bioavailability of target molecule is a key parameter. For
Sensitivity instance, targets need to be homogeneously distributed in the
FOM = sensing medium. If these targets are complexed by other
fwhm
molecules or cells, precipitated, or denatured, the ability for the
The quality factor (Q-factor) is the quotient between the sensor to detect the target is reduced. If this is the case (and
initial wavelength of a dip and the full width at half-maximum this usually happens with metal ions), the biosensing response
or minimum of this peak (fwhm). A high Q-factor indicates only detects the available fraction, which can be much lower
that the fwhm is small in comparison with its central position, than the total amount of targets present in the sample. The
so there is a high capability to distinguish small changes biological matrix or buffer itself can generate noise, so the
occurring on the surface of the sensor. WGM resonators often quality of the reagents and their filtering and degassing are
present deep peaks, with ultrahigh Q-factors.96,97 often mandatory. Moreover, the volume in contact with the
resonance
probe is important. It is the reason microfluidic systems can
Q Factor = help to increase the contact between the sensor and its targets.
fwhm All of the aforementioned parameters influence the sensing
The signal-to-noise ratio (SNR or S/N) is also often performances and need to be considered before starting any
mentioned and is inversely proportional to the fwhm. It experimental design. Each sensor must exploit its own
considers the cause of noise, such as those found in light advantages while limiting the negative influences of the
sources, absorbing medium, detectors, or other perturbations poorest parameters. This delicate balance is complex, because
of the system. But it is mandatory to be clear about the these indicators are all closely linked. This means that reaching
definition of the noise. For instance, we can consider the noise a low LOD or a high Q-factor does not mean that the sensor is
of a spectrum where resonant peaks need to be clearly reproducible or able to detect the target in real applicative
distinguished to stand out from the noise level. We can also conditions outside purified matrices and laboratory settings. It
consider the noise of a sensorgram, which in this case considers is also needed to emphasize the difference between the
the noise obtained overtime by tracking the peak/dip evolution instrumental LOD and the method’s LOD. The instrumental
within certain external medium. Clearly defined peaks/dips LOD is the most widely spread in the literature but is the one
with both low spectral noise and high Q-factor can therefore obtained with the analyte in pure conditions, as the best-case
lead to noisy sensorgrams if they are not stable. Depending on scenario and unlikely to be encountered by actual sensors. The
this, the SNR could be calculated differently. SNR is usually so-called method’s LOD is that where the analyte is measured
measured using the strength of the signal relative to the in the actual matrix of intended use (final sample) where
background noise of undesired signal and is estimated as multiple biological parameters may change the initial plan.
follows: Therefore, there is an important gap between academic
research articles and applied approaches (in the field), hence
signal level
SNR = 10 × log10 the need to standardize these detection parameters and
noise level improve the referencing of the measurements. It is also
The accuracy is the degree (%) to which the sensor readout necessary to mention the fact that many other parameters can
value corresponds to the actual value of the measurand come into play in the context of clinical and commercial
(analyte). It represents the deviation between the mean value sensors. Establishing the key parameters and increasing their
achievable from a data set and the true value of the measurand availability in research articles would make it possible to fairly
assumed as a reference value. The accuracy is different from compare sensors between different applications, under stand-
precision, as the latter represents the level of dispersion of the ardized experimental conditions and uniform data anal-
data set (e.g., the variance) with respect to its mean value. A yses.93,99−101
measurement can therefore be precise but not accurate, and
the opposite is also true.
The specificity is the ability of the sensor to recognize only
■ GENERALITIES ON BIOSENSORS
A biosensor is an integrated receptor/transducer device which
targets and not respond to other molecules or cells present in is capable of providing selective quantitative (or semi-
the medium. A number of false-positive or false-negative versus quantitative) analytical information using biological recogni-
true-positive and true-negative responses need to be high- tion elements.102 One of the most widespread examples of
lighted to obtain both sensitivity and specificity ratios. To biosensors is that used by diabetic patients. The glucose sensor
determine it, it often relies on an arbitrary threshold. If the exploiting the glucose oxidase enzyme (GOx) indeed
sensing response is over this threshold in a medium with represents more than 90% of the biosensing market.103
nontarget molecules, the sensor is not specific enough. A good However, many other types of biosensors have emerged over
balance between the noise level, sensitivity, and specificity the past decades. Among them, a wide variety operates with
needs to be set, especially in complex media. antibodies or derivatives as bioreceptors and is part of a larger
Repeatability is the ability to reproduce the same results family of immunosensors. From paper-based rapid tests to
under the same experimental conditions. It is only possible to laboratory-based assays such as the ELISA, antibodies play a
define a range of repeatability after many tests (n > 10) and is tremendous role in both sensitivity and selectivity of bioassays.
often calculated in %. While repeatability involves the same On the other hand, deoxyribonucleic acid (DNA) and
conditions (location, measurement procedure, observer, instru- ribonucleic acid (RNA) strands also allow molecules to be
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Figure 9. Artistic view of all the bioreceptors with their main assets/drawbacks. The font size emphasizes their practical use abundance among all
existing bioreceptors.

detected with high specificity and are increasingly used in rarely the case, and most strategies use a random
bioengineering. Finally, new trends in bioreceptors such as immobilization, leaving only a certain percentage of functional
biomimicking polymers and molecularly imprinted polymers sites on the surface. The size of an antibody is roughly 10 to 15
(MIPs) have been investigated in the field. The vast choice of nm and weight ∼150 kDa. It is important to take both sizes
bioreceptors and transducers sometimes makes it arduous to and molecular weights of receptors and targets into
precisely determine the advantages and disadvantages of these consideration to achieve sufficient changes on the surface
elements, which are too often blindly tested by trial and error. while binding.108 Antibodies are first choice because they are
In this section, we review these bioreceptors and provide well documented and widely available commercially and
suggestions for their use for WGM sensing. present a good affinity for their target.
Selection of an Adapted Bioreceptor. In practice, a An alternative to antibodies is to substitute them with
researcher who wishes to develop a new type of biosensor first molecules synthesized in laboratory settings. This is the case
establishes the state-of-the-art biosensor and defines a for DNA and RNA, which can be designed and chemically
molecular target. Then, the choice of a bioreceptor depends modified according to specific needs. The role of nucleic acids
on experimental conditions (nature of the sample, duration of is well-known, and they have been utilized in many fields, in
the analysis, available laboratory equipment, etc.) (Figure 9). particular for the detection of complementary DNA strands.109
Most biosensors studied for research are based on the Nevertheless, nucleic acids can be studied and selected in the
interaction between an antibody and an antigen. This antigen presence of targets of different natures (including heavy
is often a target protein, but it can also be of different nature. metals) in order to determine sequences of interest for
For example, antibodies are able to bind proteins or protein biodetection. This is the case for aptamers, which can bind to
fragments, surface proteins present on cells, other antibodies proteins or cells with high affinity and selectivity. Aptamers are
(e.g., secondary antibodies), but also lipids and toxins, among therefore described as “artificial antibodies” for this reason,
many others. Usually, immunoglobulins G (IgGs) are used as a although they have a much smaller size (up to a few
bioreceptor.104−106 They are composed of two heavy and two nanometers and around 5 to 15 kDa). Antibodies and
light chains of amino acids, connected by means of disulfide aptamers both require certain conditions to work properly,
bonds leading to a “Y-shape” structure. Both chains are especially in terms of pH and ionic composition of the
characterized by constant and variable parts, called paratopes, medium, to maintain their 3D-conformation and their key/
and are located at the end of the two branches of the antibody. lock function. Nevertheless, aptamers can deal with more
These paratopes have a strong affinity for a specific sequence severe conditions than antibodies and are therefore more
and conformation of a part of the target molecule, called the suitable for environments different from physiological con-
epitope. Therefore, a given antibody is highly specific and ditions.110−113 They are also less sensitive to these variations
selective to its corresponding epitope and is used as a (pH, hydration, temperature), which lead to a longer lifetime,
recognition element.107 Both arms of the antibodies are also especially under harsh acidic regeneration steps. They show
connected to a flexible hinge region, allowing the detection of reduced batch-to-batch variation compared to antibodies and
targets within a certain range of angles (3D detection). In are of interest in many fields, such as for targeted drug delivery.
order to optimize the binding efficiency, these regions must be Last but not least, aptamer modifications allow the addition of
oriented toward the external medium. It is therefore relevant to selective chemical groups and spacers at predetermined
bind these antibodies with a certain orientation. But this is positions, allowing substrate-oriented immobilization and
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limiting steric hindrance for an optimized binding. Despite lactate and cholesterol oxidases, horseradish peroxidase
these many advantages on paper, they still have not been (HRP), and urease are also highly important in the field.
widely used due to their lower commercial availability. The DNAzymes, also called DNA-enzymes or deoxyribozymes, are
selection process (named SELEX, for Systematic Evolution of another type of enzyme that can be involved in biosensing.
Ligands by EXponential enrichment) is tedious and expensive, They present enzymatic activities but are made of
so until sequences are not publicly available, antibodies remain oligonucleotides (RNA or DNA). Some of them are under
chosen. In terms of price, aptamers were initially more clinical trials as drugs for many diseases as they can target the
expensive at their start, but prices of antibodies and aptamers expression of certain genes. Others have been highlighted for
are now comparable. New concepts are regularly patented and their capabilities to target specific metal ions or for their
put on the market for their novel properties, such as for multiplexing with aptamers.124−127
peptimers, aptabeacons, or aptabodies, to name a few. Playing Molecularly imprinted polymers (MIPs) are synthetic
with 3D structures of DNA is also a top research interest, often receptors built in vitro against a target molecule or cell. They
called “DNA origami” allowing the formation of nanostruc- are considered analogues of natural antibody−antigen systems,
tures and nanomachinery.114−117 The future will tell if they mimicking their behavior through the “lock-and-key” principle.
finally become more competitive than traditional biorecep- MIPs are popular thanks to their polymeric nature, so they can
tors.118 be produced at low cost and stored for long times as they do
Along with the use of antibodies, the development of other not require specific storage conditions such as high freezing
binding proteins such as monobodies and affibodies has proven usually required for antibodies. They can also be produced
useful in many antibody-like applications.119−121 Among these against almost any target (from small molecules to cell
molecular alternatives, the use of camelid heavy-chain antibody surfaces). These receptors are often used in specific
fragments commonly named nanobodies (Nbs) quickly applications, but their lack of specificity is sometimes pointed
became popular. Animals such as alpacas, camels, and llamas out in the literature.128,129
produce immunoglobulins with only one heavy chain. They are A final type of bioreceptor that needs to be mentioned, but
structured in two constant regions, one hinge region, and an is least common, is the use of whole cells. Single cells or
antigen binding site. Some of these single-domain antibodies cellular tissues can be used to detect environmental changes,
can also be produced from some species of cartilaginous fishes but interactions with these types of receptors are usually less
or sharks. Nanobodies are among the shortest available specific and require a more global analysis of the detection
antibody-like bioreceptors (<3 nm and around 13 to 15 conditions and affinities. Viruses such as phages, cell
kDa). They are known for their higher resistance against heat
organelles, and parts of cellular membranes have also been
and higher stability against detergents. They also seem more
exploited and highlighted in the literature as potential
soluble in water compared to conventional antibodies. The
bioreceptors.130,131
major asset of Nbs is their ability to bind to smaller antigens
Experimental Conditions. Biosensors are usually tested
not accessible to whole antibodies. They are studied as
under ideal laboratory conditions. The most favorable uses for
bioreceptors to target small analytes or to improve site-directed
classical molecular detections are performed in water-based
immobilization techniques but they are mainly investigated as
therapeutics. Nbs are expected to cross the blood−brain buffered solutions at a constant pH and temperature. For
barrier and penetrate solid tumors more easily than conven- example, phosphate buffered saline (PBS), (4-(2-hydroxyeth-
tional antibodies, which would help for the development of yl)-1 piperazineethanesulfonic acid) HEPES buffer saline
new targeted therapies and for diagnostic strategies.122,123 (HBS), or tris(hydroxymethyl)amino-methane (Tris) are the
They are used as sensing elements due to their ability to most common solutions, as they maintain a stable pH between
selectively bind to target molecules, such as proteins or small 7 and 8 and mimic physiological osmolarity and ion
molecules, with high affinity and specificity. In WGM sensors, concentrations. Nevertheless, their composition and concen-
nanobodies could be investigated and act as the recognition tration can vary, in particular, by the addition of low
elements for existing techniques relying on antibodies. concentrations of surfactants such as detergents added to
Enzymes are also involved in many biosensors. They can be increase the surface wettability and the contact between the
described as a group of molecules produced by living surface of the biosensor and the sample. Working with freshly
organisms that cause biochemical reactions. They are present prepared and degassed buffer is often a key to obtaining
in all cells and can be secreted, as is the case, for example, in reliable results. Many factors can influence the reproducibility
saliva. Enzymes play essential roles in the living world and are of the experiments and the affinity between receptors and
the key to all metabolic processes. Most enzymes are proteins, targets, especially those affecting their structural integrity. First
but they can be made of ribonucleic acids (RNA, called of all, the storage conditions need to be adapted depending on
ribozymes) or involving other macromolecules. Enzymes are a short term (few weeks) at 4 °C or long term (from month to
catalysts, so they enable reaction processes by increasing their years) at −20 °C to −80 °C. It is also recommended to use
speed (lowering the activation energy) without appearing in cryoprotectants such as glycerol to avoid the formation of ice
the final reaction balance. However, they differ from other crystals, affecting the protein structures. These additives are
chemical catalysts in their high efficiency and specificity. They often already present inside commercial samples.
are able to catalyze, chemically recognize, bind, cut, or modify Working with refractive index changes, such as with optical
substrates and are often extracted from cells to enable resonators, at different concentrations diluted from these stock
commercial processes. They are, for instance, used in many solutions can therefore lead to small signal changes that are not
cleaning and washing products, play the role of analytical related to detection but simply to differences in stock
devices, and have clinical and environmental applications, conditioning from the experimental conditions. It is also
among so many others. Glucose oxidase (GOx) is one of the highly recommended to aliquote stock solutions in order to
most common examples in biosensing, but others such as avoid freeze/thaw cycles.132,133
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Figure 10. Different coupling chemistry for the surface biofunctionalization of WGMs. Strategies for adsorption, covalent binding, orientation of
the receptors, and practical tip and tricks.

Interactions between the target of interest and the receptors all the biochemical conditions with standardized techniques
need to be verified using other standard laboratory techniques. makes it possible to avoid any doubt concerning the targeting
It is indeed often needed to validate the biomolecular couple strategy. Techniques such as fluorescent microscopy, atomic
before starting the experiments on a new platform to ensure force microscopy, quartz microbalances, prism-based SPR
sufficient binding efficiency. It also decreases uncertainties by devices, Western blots or dot blots, and ELISA or PCR tests
changing different experimental parameters at a time. Testing are some methods to consider verifying the presence of an
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analyte in a specific sample or to check the binding upon a molecules and the −OH surface groups. Modified silanes
surface. Last but not least, performing controls (with or such as mercapto-, amino-, or expoxy- can also provide a strong
without the target, with or without bioreceptors, with other anchor to move the process further and enhance the reaction
nonspecific molecules) is essential to demonstrate the effect. It is therefore important to finely control the deposition
specificity and relevance of the technology. To do so, it is parameters such as the incubation time and concentrations of
often necessary to adapt surface blocking steps and to adjust reagents and temperature, enabling the formation of a self-
the experimental conditions (concentrations, incubation times, organized layer. Different types of silanes are commercially
and rinsing) to optimize and ensure sufficient surface control. available, often differing by minor changes on their molecular
The purpose of surface blocking is indeed to prevent chains or groups. The binding through silanes is chemically
nonspecific adsorption of unwanted materials on the surface, stable and shows relatively low levels of steric hindrance, as
leading to false positive detections. BSA, powder milk/casein, they are sufficiently distant from the surface to bind to the
or fish gelatin are commonly used blocking agents to be target receptors. Furthermore, it is important to mention that
adsorbed onto the surface of a material and block nonspecific an excessive concentration of silanes or an exceedingly long
interactions. These are, for example, often used in ELISAs as deposition time can lead to the formation of agglomerates and,
well. thus, decrease the quality of the sensor surface. Drying and
As an example, it is not uncommon to encounter batches of heating the surface in an oven after silanization is also often
antibodies that do not deliver the expected affinity, and the studied as helping to form a more uniform layer. Some well-
variability from one supplier to another can be significant. known silanes used for biofunctionalization are (3-
Their production originates in animals but is carried out via aminopropyl)triethoxysilane (APTES), (3-aminopropyl)-
cell cultures and purification steps which may vary over time, trimethoxysilane (APTMS), and 3-mercaptopropyltrimethox-
although these processes are increasingly reliable and better ysylane (MPTS).141−145
controlled.134 Batches labeled “for research use only” (RUO) In addition to surface modifications, other techniques can be
can sometimes lead to less efficient quality tests compared to used to spot DNA on such surfaces. The use of modified DNA
those for clinical/industrial uses, but these are often the only sequences such as silanized nucleic acids (with embedded silyl
option available to research laboratories. More than 6000 groups) can remove the need for glass modification. Molecular
commercial antibodies were tested independently from 26 nanoprinters used in the DNA microarrays industry could also
suppliers. More that 75% of these antibodies were nonspecific be investigated in this case to coat the WGMs surfaces with
or did not work at all.135 The Human Protein Atlas consortium high density by a localized stamping mechanism. Bioreceptors
also examined more than 5400 commercial antibodies from 51 including DNA-aptamers can therefore be covalently immobi-
providers, from which more than 50% could not be used for lized on both silica and silicon through these processes.146
their anticipated application.136,137 Biological compounds do Aside from nucleic acid-based receptors and their deriva-
not allow for their blind use on a sensor. Many control steps tives, proteins such as enzymes and antibodies can be
must be carried out to verify all the process steps, from their immobilized thanks to the famous NHS/EDC reaction. It is
affinity validation to their immobilization on the sensor’s built on the carbodiimide reaction, calling on N-hydroxysucci-
surface.138,139 What is true for antibodies is probably also true nimide (NHS) and (dimethylamino)propyl)carbodiimide
for other types of bioreceptors. It is therefore mandatory to be (EDC) intermediates to anchor an appended carboxylic acid
aware of the importance of the reagents and check their quality group of the receptor to the amine group of a silane. The
at all stages of the long path, leading to the development of a opposite reaction is also possible, both leading to the
reliable biosensor. formation of a covalent amide bond (named a peptidic

■ SURFACE FUNCTIONALIZATION
Immobilization on Silica and Silicon Substrates. Most
bond). This reaction is also possible with DNA, peptides, and
other compounds. The use of NHS/EDC is interesting
because it is biocompatible, has a very little influence on the
WGM sensors are usually made of silica (SiO2) or pure silicon bioactivity of target molecules and leads to cleaner products
(Si), an inert material that is resistant to important chemical or than other cross-linkers that could also be used to perform
physical constraints and maintains its integrity when it enters surface chemistry, such as formaldehyde and glutaralde-
into contact with water or physiological buffers. Silica is a hyde.147−149 Epoxy-silanes are less used, but they can react
widely used substrate in biochemical assays, especially to with thiols or amino groups by a direct interaction. Many types
manufacture DNA chips. To allow the adhesion of of silane molecules and reactive groups can lead to strong
bioreceptors on glass (covalent bonding or adsorption), anchoring of a biolayer, depending on its own structure and
many strategies are available (Figure 10). First, the surface the ability to chemically modify the target receptors.
needs to be thoroughly cleaned and activated. Covalent Well-known interactions such as those between avidin (and
bonding is then often performed through silanization by derivates) and biotin (vitamin B8 or H) can be exploited as
organosilanes and using cross-linkers, while adsorption is often well. Receptors can usually be conjugated with biotin
physically enhanced by exposing the glass surface to UV/O3.140 molecules to bind to any avidin coating through available
The silanization process is based on the transformation of Si− commercial kits. In this case, a surface coupling of avidin/
OH groups into reactive species. To do so, it is necessary to streptavidin such as for NHS/EDC chemistry is perfectly
thoroughly clean the silica surface and increase the amount of suited. Once the target is biotinylated, this method remains a
−OH groups on the surface by immersing the silica/silicon classical, polyvalent, and functional technique to immobilize
substrate into piranha acid (H2SO4 and H2O2, 3:1). This step bioreceptors.150,151
is crucial in order to remove all dust and impurities from the Immobilization on Gallium Nitride (GaN) and Indium
surface after the fabrication of WGM sensors. After that Nitride (InN). Gallium nitride is not a common material used
thorough surface cleaning, the immersion in silanes leads to in bioassays. However, it is well-known in the WGM
strong interactions (siloxane bonds) between the silane community. Recent studies have shown that GaN is
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biocompatible and fits with biosensing applications. In this adsorption of bioreceptors can also be considered as an
context, molecules such as peptides have been immobilized on alternative.162
GaN surfaces using thiols and PMPI to link a cysteine residue Immobilization on PDMS. Polydimethylsiloxane (PDMS)
to the −OH end of thiol molecules, and the nontoxic effect of is a widely used polymeric material, especially for specific
the material has been tested on cell cultures.152 GaN is WGM packaging. It can be implemented in its liquid phase and
established as an excellent material for lightning and lasing polymerized on site to protect and carry structures such as
applications in the visible spectral range. It has also been taper fibers or microspheres. PDMS is usually cured at room
studied for the excitation of quantum wells positioned at the temperature for 1 day or in moderate temperature (∼60−80
surface of a microphotoluminescence setup using HRP. The °C) for 30 min. It is optically transparent and flexible and is
microfluidic channels used in this particular study were easy to handle.163 For biosensing purposes, its hydrophobicity
covered with a polydimethylsiloxane (PDMS) membrane is an issue and needs to be decreased, as silicones can be
hydroxylated using oxygen plasma and functionalized using difficult to integrate with other substances because of their low
aminopropyldimethylethoxysilane (APDMES) 1 vol % in surface energy. To create a hydrophilic surface, polar groups
isopropanol. The surface was then heated to 85 °C for 30 need to be added by using a plasma or UV treatment. Hydroxy
min. Indium nitride has also been used to build new types of groups (−OH) can also be chemically added using H2O2/HCl
biochemical sensors. The surface functionalization relies on the (or piranha solution, using H2SO4), NaOH, KOH, etc. Once
same strategies as those used to functionalize silica or silicon, activated, the PDMS layer needs to be functionalized in the
through silanization or coating-layer formation.153 following hours, as the surface tends to return to its
Immobilization on Titanium Dioxide (TiO2) and equilibrium state through the diffusion of oxidized low
Lithium Niobate (LiNbNO3). Titanium dioxide (TiO2) molecular weight chains, reducing surface energy. It is also
coatings belong to the family of transition metal oxides and possible to implement poly(ethylene glycol) groups (PEG)
are biocompatible and environmentally friendly. It is important through vinyl-terminated PEG molecules using hydrosilylation.
to mention that organic or inorganic hybrid forms of TiO2 Among the many strategies available to covalently functionalize
materials exist specifically to improve sensing properties and PDMS layers, click chemistry using thiols remains one of the
are currently considered a hot topic. To its origin, titanium fastest process. Mercaptosilanes such as (3-mercaptopropyl)-
(Ti) and alloys were already recognized as biocompatible trimethoxysilanes (MTS) in methanol is commonly used.164
materials for biomedical devices such as for implants, although However, several techniques can be implemented to provide
side effects can sometimes be noticed. Ti can be modified different surface groups. For instance, it is possible to
chemically/physically in TiO2 to further improve its bio- functionalize PDMS with biotin groups by adding biotinylated
compatibility and promote surface molecular absorption, phospholipids directly to the PDMS before curing.165 APTMS
playing a crucial role for tissue regeneration.154 In the context and APTES silanes, followed by NHS/EDC chemistry, can
of WGM resonators, TiO2 is often exploited for its moderately lead to covalent binding of proteins and antibodies, as
high refractive index (2 to 2.7 depending on its phase), large previously described on silica surfaces. However, slightly
bandgap absorbing light below 390 nm, and low material loss higher concentrations of silanes can be used with shorter
from visible to infrared wavelengths. TiO2 is also chemically incubation times to avoid swelling and distortion of the PDMS
stable, rigid, and robust, which makes it a good candidate for layer.166 Other original approaches are based on polymer
CMOS and nanofabrication processes, such as for on-chip grafting, which can rely on “grafting from” or “grafting to”
WGM resonators.155 Different TiO2 configurations have been strategies. “Grafting from” means that radical groups present at
developed, especially nanohybrid layers consisting of mixing the PDMS surface are used to initiate the polymerization of a
both materials and bioreceptors to strengthen their binding desired polymer. The addition of a photoinitiator followed by
and forming a new material named “mongrel”. This resulting UV treatment are usually performed. The “grafting to”
organic or nonorganic nanomaterial can be covalent or technique means covalent binding of polymers to the
noncovalent, using hydrogen bonds, van der Waals forces, or functional groups added on the PDMS layer such as amine
electrostatic forces, as a novel and original functionalization. or thiols. Last but not least, blocking agents such as BSA,
More conventional techniques relying on the formation of a gelatin, or PEG molecules can be added and provide a good
self-assembled monolayer (SAM) using hydroxyl groups balance between wettability and antibiofouling properties.167
(−OH) by oxygen plasma exposure and/or piranha solution More exhaustive and specific biofunctionalization processes
are also described in the literature. Common protocols for can be found in dedicated biochemistry textbooks such as
antibodies or protein immobilization rely on APTES Bioconjugate Techniques,168 Surface Modif ication of Polymers:
monolayers followed by a glutaraldehyde cross-linking.156 Methods and Applications.169 and review articles.170−173
Nucleic acid-based receptors can follow the same biochemistry Biosensing Enhancers. To achieve accurate detection of
or use original linkers such as with polylysine (PL) molecular binding events, it is essential to reach both high
intermediates157 or through weak bonding (adsorption), with specificity and high sensitivity. However, when characterizing
geometry-dependent mechanisms, as highlighted in Silva- the bulk refractive index sensitivity of a sensor using solutions
Moraes et al.158 WGMs with TiO2 layers have been tested with high index jumps (such as salt- or glucose-based
under different biosensing formats, recently in the frame of solutions), it is difficult to make a fair comparison with the
SARS-CoV-2 detection.159 Another material of interest is ultrasmall refractive index changes that occur when molecules
lithium niobate (LiNbNO3).160 It is usually used as a bind to the surface. This is why it is important to optimize the
piezoelectric substrate to build acoustic sensors. Its function- surface properties, ensure low nonspecific binding, and
alization through amino-silane and cross-linking has been improve the signal-to-noise (SNR) ratio of the sensor.
performed and validated with good binding capacity. The use Additionally, researchers may use signal amplification
of protein G intermediates has also shown an enhanced techniques such as functionalized nanoparticles to enhance
sensitivity in previous immunosensing works.161 The direct the signal from the binding event. These switches the sensors
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Figure 11. Scheme of different amplification strategies from the sandwich assay, fluorescent amplification, enzymatic reaction, nanoparticle
amplification, or coupling agents.

from “label-free” configurations to “enhanced” or “tagged” nonspecific adsorption, generally always higher and almost
configurations. inevitable at basal levels with this kind of approach. It is
Bulk refractive index sensitivities are often characterized with therefore necessary to ensure sufficient relative signal to get out
salt or glucose solutions prepared with high index jumps, of the noise level and certify the detection even at low
leaving little room for a fair comparison with ultrasmall RI concentration levels. Abundant rinsing, buffer stabilization, and
changes occurring on the sensor while molecules are binding filtering can be essential steps to avoid such effects on the
upon its surface. To increase the impact of these interactions, surface. Finally, other strategies such as those exploited for
especially at low concentrations, physical enhancers such as DNA hybridization could be investigated on WGM sensors,
metallic nanoparticles (e.g., the ones made of noble materials such as double-strand markers, enabling the detection of DNA
for surface plasmon resonance, localized surface plasmon or RNA hybridization with their complementary sequences.
excitation, or for surface enhanced Raman spectroscopy),174,175 Techniques such as bioluminescence resonance energy
secondary binders or fluorescent tags can be added.176 This transfer (BRET) and Fö rster resonance energy transfer
signal amplification is far from being a weakness for a (FRET) can be adapted to WGM resonators. BRET and
technique because it clearly improves the detection threshold FRET are based on donor and acceptor molecules that are in
and its reliability, while allowing secondary control as is the close proximity, so the donor absorbs energy from the
case with fluorescent labels, for example. The first and simplest excitation source and transfers the energy to the acceptor,
technique to implement is the addition of secondary antibodies which in turns emits a detectable signal.177 These techniques
in order to amplify the detection by a mass effect over a target are commonly used in biology with fluorescent/biolumines-
molecule. Often called “sandwich assay”, this method is for cent proteins as donors.178 They have been broadly used to
instance a key enabling strategy in ELISA (Figure 11). While visualize intracellular activities and to determine signaling
monoclonal antibodies can bind to one epitope of the target pathways such as those involving protein kinases and small
molecule, polyclonal antibodies can bind to several epitopes of GTPases (family of hydrolase enzymes that bind to nucleotide
a single target. It is therefore possible to multiply this effect on guanosine triphosphate). Fluorescence lifetime imaging
a single binding event using polyclonal secondary antibodies to (FLIM) or fluorescence lifetime photometry (FLiP) are
bind to multiple sites available on the trapped targets. The use other interesting features that could be exploited to monitor
of chromophores/fluorophores on these same antibodies is molecular activities over time.179,180 Exploiting this technology
also possible and enables the validation of the binding through on WGM could determine the positioning of certain molecules
the fluorescence level. This is particularly relevant in research within cells or validate the capture of specific receptors and
to validate the binding protocol. However, fluorescence does their analytes. It is known that the chemical addition of small
not necessarily bring information, especially while working at fluorophores on receptors or targets does not significantly
typical wavelength ranges that do not fit with the tag windows. affect binding capabilities, so the affinity remains high. On the
An overlap is also often challenging to manage, especially with other hand, luminescent semiconductor nanocrystals (also
the narrow wavelength ranges usually used for WGM named quantum dots, QDs) possess unique features for
interrogation. The use of metal or polymer nanoparticles biosensing. They are roughly spherical particles ranging from 1
then takes full meaning, especially because they cause an to 12 nm and exhibit different optoelectronic properties from
increase in the local refractive index shift and can also cause continuous solid layers. These properties can be tuned by their
light scattering under certain conditions. Their biofunctional- size and shapes, and they present high photostability, narrow
ization and surface preparation is therefore needed to avoid fluorescent emission, and broad absorption spectra. QDs have
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Table 1. Nonexhaustive List of Recent Achievements in WGM Biosensing

WGM structure Analyte (molar mass) Bioreceptor Achieved LOD Reference
Microbead Cytochrome C (∼12 kDa) Antibody (biotinylated) 6.82 × 10−9 M Chen et al., 2018193
Microcavity Horseradish peroxidase (HRP)/VEGF Antibody 0.3 ng/mL and 17.8 fg/mL Ouyang et al.,
(∼40 kDa) 2020194
Microdisks Bacteria (Staphylococcus aureus) LysK (phage protein) 5 pg/mL (∼20 cells) Ghali et al., 2016195
Microdroplet (liquid Copper (Cu2+) (63.5 u) Stearic acid 40 pM Duan et al., 2019196
crystal)
Microdroplet (liquid Urea (60.06 g/mol) Urease/stearic acid 0.1 mM Duan et al., 2020197
crystal)
Microsphere Mercury (Hg2+) (200.59 u) Aptamer (DNA sequence) ∼50 nM Wu et al., 2016198
Microsphere Glucose (180.15 g/mol) Glucose oxidase enzyme (GOx) N.A. Brice et al., 2020199
Microsphere Labeled antibody Antibody N.A. Giannetti et al.,
2012200
Multiple Microspheres CA-125 cancer biomarker in serum (3−5 Antibody ∼1.8 U/mL (arbitrary medical Huckabay et al.,
MDa) unit) 2013201
Spherical microparticles Protein (cell surface proteins) Antibody (immobilized through ∼0.5 nM Freile et al., 2021202
Protein G)
WGM Fiber Streptavidin (∼60 kDa) M13 bacteriophage 3 nM Hsieh et al., 2022203

been a hot topic and highly studied over the last 15 years but scales with √ε due to the unique eigenfrequency topology and
probably remain a key technology to build new types of can be greater than the linear response at DPs.185,186
fluorescent biosensors. Their implementation presents a high
interest for research but their practical use is still limited due to
their high cost and the use of toxic solvents (especially for
■ FREQUENCY COMBS
Frequency combs are equidistant spaced frequency compo-
those working at higher wavelengths), not always suited for
nents that can be generated across the infrared, visible, and
biosensing applications.181,182
ultraviolet spectra of light.187 Previously, frequency combs have

■ EXCEPTIONAL POINTS
Open systems in optics with gain or dissipation are described
been generated by intracavity phase modulation. Kippenberg et
al. showed a new approach to generate “micro” frequency
combs based on WGM resonators. Here, a continuous wave
by a non-Hermitian Hamiltonian matrix (a matrix describing laser with high power (tens of mW) coupled to a WGM
the energy of the system). For conventional non-Hermitian microresonator is able to generate optical intensities strong
WGM resonators that operate at diabolic points (DPs), the enough to induce Kerr nonlinearity which subsequently
corresponding eigenvectors are orthogonal, even for modes generate sideband components forming a frequency comb.188
with identical resonance frequencies (e.g., clockwise and Taking this work further, Vahala et al. utilized dual comb
anticlockwise modes). In this case, the system’s sensitivity to technology and the concept of optical interference for
perturbations, quantified as the difference of eigenfrequencies spectroscopy. The researchers designed a typical interferom-
Δω, is directly proportional to the strength of the perturbation, eter with a reference arm and a sample arm. By looking at the
ε. (that is, Δω ∝ ε). However, when the non-Hermitian WGM interference pattern of each comb from each arm of the
resonators are tuned to what is called an “exceptional point” interferometer, spectroscopy could be realized.189 This field
(EP) where the eigenvalues and eigenvectors coalesce, there is continues to evolve every year. In 2023, Jun Ye at the
a drastic change in the response of the resonators to small University of Colorado, Boulder, showed a novel breath
perturbations. The advantage of tuning a WGM resonator to analysis of the SARS-CoV-2 virus using cavity-enhanced direct
an EP is to enhance the platform’s sensitivity to perturbations frequency comb spectroscopy. This state-of-the-art technique
(e.g., the perturbations from nanoparticles or molecular is capable of generating a large amount of molecular absorption
binding).87 Once the non-Hermitian WGM resonator is features in real-time, getting down to part-per-trillion volume
tuned into an EP, the sensitivity to perturbations shows a sensitivity.190 The use of molecular absorption features can
dependence on Δω ∝ ε1/N. Since ε 1/N ≫ε for sufficiently help to improve the specificity of the WGM sensors.
small perturbations (ε → 0), the sensitivity at EPs can be
enhanced compared to that at DPs. In the case of DPs, there is
no intrinsic backscattering without nanoparticles and therefore
■ RECENT BIOSENSING ACHIEVEMENTS USING
WGMS
no mode splitting. When nanoparticles enter the mode volume WGM sensors have been widely studied for their biosensing
of the resonator, mode splitting occurs due to backscattering at potential. Some of them are capable of reaching ultralow limits
the nanoparticle location.183,184 of detection up to cutting edge single-molecule levels usually
To induce a second order (N = 2) EP in a single WGM achieved under plasmonic configurations. Using specific
resonator, one approach is to use two silica nanotips to tune combinations of WGM structures and surface biochemistry,
the scattering between the clockwise and anticlockwise modes. it is possible to achieve the detection of DNA, ions, particles,
Exciting an EP requires precision or external off-chip and viruses but also proteins and cells. Some of these recent
components (nanoscatterers such as nanotips). The EP is biosensing achievements are listed in Table 1. Among these
achieved when the tuning of the nanotips results in the full works, innovative detection strategies have been described and
asymmetric backscattering of the two counterpropagating tested, in particular, for the detection of bacteria such as
modes. Therefore, when a target nanoparticle enters the Staphylococcus aureus or Helicobacter hepaticus. In order to
mode volume of an EP sensor, the mode splitting Δω now detect binding events such as the ones obtained for single-
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Figure 12. (a) Scheme of a WGM microlaser-integrated optofluidic chip for ELISA. The microfluidic chip contains: eight inlets for injection of
different reagents; microchannels for delivering reagents and integration of WGM microlaser sensors; one spiral mixer for mixing reagents;
fishbone-shaped grooves for embedding of optical fibers; one outlet for collection of liquids after reaction. (b) Workflow of in situ modifications of
the surface of a WGM microlaser with functional molecules for on-chip optofluidic ELISA of VEGF. Reproduced with permission from ref 194.
Copyright 2020 The Royal Society of Chemistry.

molecule sensing, the surface of the WGM itself usually needs cell cultures or protein batches could also rely on their high
to be functionalized with other molecules or nonorganic sensitivity. For instance, the monitoring of ultrasmall forces
particles to improve the sensing modalities. For instance, and strains within or between cells could lead to a better
coupling metal nanoparticles on WGM microcavities is a understanding of their metabolism, growth, and communica-
method that enables the detection of molecules with small tion. WGMs could also be used to track molecules in real time,
polarizabilities. This has been demonstrated on single-strand probably using specific labels and tags to enable their
DNA (ssDNA).191 This being said, sensing devices down to monitoring over time and under molecular imaging in the
single-molecule levels are sometimes considered as the 3D space.
ultimate tools in research, to the detriment of other parameters On-Chip Biosensors. One of the limiting factors of WGMs
such as reproducibility and ease of use, which are key remains their practical integration. However, some structures
parameters in field applications, such as medical diagnosis. We such as microrings or microtoroids are well suited to be
can easily question the way in which a single-molecule packaged and directly integrated inside microfluidic chambers,
detection would be possible in common practice and how to for sensing in both aqueous and gaseous media. Some
reach enough specificity to ensure targeted detection.192 In microfluidic integrations have been recently designed and
most cases, “single-molecule” sensing is only obtained implemented using microtoroids for immunosensing, as
statistically or using larger nanoparticles embedding hundreds reported in Gorodetsky et al.194 (Figure 12). In this work,
or even thousands of molecules to detect in order to achieve a the authors show transposition of the ELISA technique on the
sufficient signal shift and be experimentally viable. It is microtoroid substrate for HRP-streptavidin and VEGF
therefore important to contextualize the manipulation not to detection. The sensitivity experimentally reported is 2 orders
mislead the reader with too optimistic theoretical LOD values. of magnitude lower than with conventional ELISA tests, which
On the other hand, it is clear that the sensitivity of WGMs is a remarkable performance.
would make it possible to identify molecular or even Microsphere Biosensors. These on-chip-manufactured
intramolecular events such as, for example, the change of structures contrast with the use of microspheres and taper
conformation (folding) of a protein or mutations related to a fibers, which are the least expensive in terms of production and
single nucleotide in DNA or RNA sequences. The detection of easier to handle in terms of light coupling thanks to the taper
polynucleotide strands at such low levels would also avoid the fiber, in laboratory settings. The latter can be easily
need for PCR amplification cycles and save significant time manufactured from single-mode fibers and a heat source, in
compared to the current routine techniques. comparison to on-chip structures requiring several steps and
Ex vivo and in vivo applications are also key points, although equipment, as explained in the section on On-Chip
integration of WGMs and related standardized methods, such Resonators. The use of substrates often leads to coupling
as for sterilization, are often required to work in biomedical issues and requires precisely designed adapters to avoid
environments. Aside from direct biodetection, the use of significant signal losses for their integration. Microspheres
specific responsive coatings could be investigated to monitor interrogated with taper fibers bypass this issue using a direct
humidity and pH. This idea often comes up to study the connection through the optical fiber from the laser to the
evolution of a disease or a severe injury, by integrating these detector. However, the coupling between the microsphere and
sensors under bandages and plasters. In vitro applications in the extremely fragile region of the taper fiber (only a few-μm-
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Figure 13. (a) Scheme of the typical experimental setup used with microspheres WGMs. The microsphere is produced at the tip of an optical fiber
and placed perpendicularly to the thinner region of a taper fiber in order to excite the WGM and couple light back to the detector. (b) Graph
showing the WGM response during the functionalization of the microsphere (estimated mass loading in pg/mm2). (c) Graph showing the
detection of Hg2+ ions on the functionalized microspheres. (d) Scheme of the functionalization strategy for Hg2+ detection using DNA aptamers
through strand displacement reaction. The cDNA is attached first using streptavidin on a biotinylated dextran matrix and the aptamer is then
hybridized. Reproduced and adapted with permission from ref 198. Copyright 2016 MDPI (CC-BY).

Figure 14. Schematic illustration of the orientational transition of stearic acid-doped 5CB microdroplets induced by changes of 5CB anchoring at
the LC/aqueous solution interface: (a) without HM ions at pH = 6.5; (b) without HM ions at pH = 8.5; and (c) with HM ions at pH = 8.5.
Reproduced with permission from Duan et al.196 Copyright 2019 Optical Society of America.

thick silica) remains critical. While mode coupling remains factor. Although many studies have demonstrated the
convenient in air, it is very difficult to stabilize when it comes feasibility to use these structures under laboratory settings, in
to immersion in drops of liquid. Adding successive drops of particular to detect Hg2+ in solution using DNA aptamers
samples to perform functionalization and detection often (Figure 13),198 there is still room for improvements in order to
causes movements of both microsphere and taper fiber, which stabilize and improve the experimental structure, without
causes a change in the monitored signal (and additional affecting the coupling quality and ease of production. For
coupling loss) and, consequently, a deterioration of the Q- example, the use of taper fibers embedded in high refractive
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Figure 15. (a) Schematic of the PS microcavity with QDs and dyeNP immobilized on their surface. (b) Absorption spectrum of the QD 525
(dotted), emission spectra of QD 505 (black) and QD 525 (blue), and absorption (green dashed) and emission (purple dashed) spectra of the
acceptor dye NPs. (c) Energy transfer efficiencies as a function of WGM wavelength for different samples of microspheres, with the same amount of
QD 505 emitters but different amounts of dye NPs. Reproduced and adapted from Jana et al.207 Copyright 2020 American Chemical Society.

index materials or within PDMS molds (packaging) could In Oki et al.,205 the authors present a FRET-mediated sensor
improve the robustness of the technique and reduce its based on blended conjugated polymer microspheres leading to
sensitivity to movements related to the injection of samples. It the NIR emission from a WGM cavity. The two polymers used
is also question of surface tension of the liquid used, which in this study act as energy donor and acceptor and display red-
often causes a high physical effect on the taper fiber. Surface to-NIR signals in solution but not in the solid state due to
tension is often reduced by enhancing the surface wettability or aggregation quenching. HOMO and LUMO energy levels of
by adding detergents at a low concentration in the samples. A both polymers represent their highest occupied molecular
good balance must be found between the use of detergents so orbital and lowest unoccupied molecular orbitals, respectively.
as not to affect molecular detection and avoid any denaturation When light of a high enough frequency is absorbed by an
of proteins or analytes in solution. electron in the HOMO, it jumps to the LUMO, and the size of
Microdroplets and Micro/Nanoparticle-Based Reso- the gap between HOMO and LUMO drives the color
nators. About ion detection strategies, it is clear that the use produced in solution. The authors conclude by the fabrication
of WGM sensors could help for monitoring heavy metals of WGM microresonators using a self-assembly of energy
donating conjugated polymer doped with a small portion of
(mercury, copper, cadmium, lead, etc.) at low concentration.
the energy accepting polymer, which is NIR-emitting. Due to
The most common techniques to detect metal ions are based
the energy transfer (FRET) between them, a photoexcitation
on absorption, reflection, or luminescence spectroscopy. They
region appears at the perimeter of microspheres, and the
usually need long sample preparation or dyes to be observed Q-factor was around 600 in the NIR region where
experimentally processed in bulky equipment. Miniaturizing the absorption loss by the energy-accepting polymer was
an optical technique using WGMs and improving the surface almost dismissed. The idea to use quantum dots (QDs) as
specificity through functionalization is an important area of enhancing strategies has also been demonstrated through
research. The most recent literature often calls upon the CdSe/ZnS QDs embedded within polystyrene microspheres
particular structures of aptamers and DNA-based receptors to and using continuous wave excitation. The adsorption of
trap target ions. Nevertheless, several strategies have high- proteins was detected thanks to the change in refractive
lighted the possibility to achieve this objective, such as the use index.206 More recently, the use of spherical resonators to
of fatty acids.196 Using stearic acid microdroplets, researchers perform energy transfer from the cavity resonance modes
have pinpointed the detection of copper ions at 40 pM, 6 (WGM) to outside optical acceptors for sensing has been
orders of magnitude lower than the threshold defined by the performed in Jana et al.207 The authors fabricated colloidal
world health organization (31.5 μM). The study shows that CdSe-based core/shell quantum dots QDs and achieved high
spherical stearic acid droplets at certain pH (modifying their Q-factor resonances (>4000) (Figure 15a). Dye nanoparticles
properties from planar to homeotropic anchoring) and the (dyeNPs), made of rhodamine B octadecyl ester with
observation of their related pattern can be used for tetrakis(pentafluorophenyl)borate (R18/F5-TPB), were pro-
reproducible detection of heavy metal ions (Figure 14). duced and deposited on the QD filled polystyrene beads
In addition to the label-free configurations mentioned above, (Figure 15b). The absorption spectra were acquired using
a significant gain in response signal and specificity can be different sample concentrations (Figure 15c) and showed that
obtained through the use of molecular dyes and labels. The the surface sensitivity was greatly enhanced by the Q-factor of
most important feature of fluorescent resonators (besides their the cavity. The energy transfer was efficient over greater
geometry) is the gain medium, providing the required distances (>100 nm) compared to typical distances ∼10 nm
fluorescent emission that is then modulated by the WGM usually recorded for FRET. This principle could therefore be
resonances. Fluorescent dyes are the most common gain media further developed to monitor biosensing events or cellular
used, providing a broad range of emission bands. They can be events, as usually targeted for FRET sensors.
directly incorporated within a polymer such as PDMS, SU8, or
PMMA to fabricate the WGM structure204 or added on its
surface to tune the biosensing labeling through chemical
■ SINGLE-MOLECULE DETECTION
Among the recent applications of WGMs, the possibility to
moieties. FRET/BRET or QDs can also be implemented on detect analytes at ultralow concentration up to single events
the WGMs surface to provide signal enhancements (see remains an ambitious objective as well as a burning scientific
Biosensing Enhancers section). debate. While several top-cited papers emphasize the detection
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Figure 16. (a) Six-channel microfluidic chip containing 1000 wells/channel of slightly larger dimensions than the sensor beads was used. Wells
were filled with the sensors by flushing in an excess of functionalized particles. Particles not confined in wells were removed by flushing the channel
with water at flow rate of at least 40 μL/min. The laser beam interrogated each particle one-by one, and measurements were performed with a
spectral resolution of 6 pm. (b) Data analysis. Binding of molecules induced a shift in the resonance frequency of the spectrum. An average shift was
calculated for all Δλ values obtained on the 30 selected particles for each binding step. Reproduced with permission from Alvarez Freile et al.202
Copyright 2021 The Authors(s), Elsevier B.V. (CC-BY).

of single molecules, these are often limited to expectations experimentally verify that the signal shifts observed are only
from theoretical calculations or are countered by opposing due to a single particle detection and not from any other
publications, as was the case in 2007 and 2010.87,208−211 More experimental disturbance such as a slight change in the buffer
than ten years after these pioneering results, the subject composition (and subsequently its refractive index) between
remains an open discussion, especially while “biosensors” are the initial one used as reference and the one supplemented
often only theoretically presented through simulations, without with particles or viruses, which may depend on their
any experimental data. This statement is true for all types of production. These recent works are therefore at the cutting
biosensors and is not restricted to the WGM community. edge of technology and must be carefully analyzed and
Despite this ascertainment, many inspiring approaches can be confirmed, which will probably be a large part of future
highlighted in recent developments of highly sensitive WGMs. achievements over the next years.
The Vollmer group largely contributed to this field and
published many works of interest in this direction. As
mentioned in Yu et al. in 2021,186 most of these configurations
■ TOWARD PRACTICAL APPLICATIONS
As previously mentioned, the most widely used biosensing
rely on opto-plasmonic structures which are among the most strategy relies on immunosensing mimicking the ELISA
sensitive platforms to achieve these accomplishments. process where antibodies or proteins are immobilized on a
Although this research area is very promising and impressive microtiter plate and brought into contact with several samples
for the challenge of reaching such detection limits, it is clear (or sample dilutions). The use of protein G was studied by
that applications are not yet defined, and both reproducibility Á lvarez Freile et al.202 in 2021 to orient antibodies (IgGs) on
and reliability of such detections can still be questioned. It is, the surface of WGM sensors and use their full performance
for instance, difficult to find a complementary way to thanks to the available binding sites. In this work, the authors
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Figure 17. (a) Illustration of a sensor-based tissue chip. Cells are placed on top of a membrane enabling the diffusion of biomarkers, reaching
microring resonators through microfluidics. (b) Design of the chip, (c) picture of the chip after dicing, and (d) picture after antibody spotting. (e)
Scheme of the functionalization chemistry using succinic anhydride-bearing silane. (f) Antibody layout showing the different target molecules for
each ring resonator. Reproduced with permission from Cognetti et al.214 Copyright 2023 The Royal Society of Chemistry.

present the use of fluorescent microbeads of 10 μm diameter bound specifically to the immobilized biotin receptors,
working as optical resonators covered with protein G and including appropriate washing steps to measure only bound
oriented IgGs. The binding between receptors and targets streptavidin. The researchers show improved sensitivity to
provides resonance shifts allowing a versatile 3D assessment of biomolecule binding compared to silicon ring resonators by
binding characteristics. The NHS/EDC strategy was used for creating a slot-ring waveguide and by utilizing the low
biofunctionalization, and a six-channel microfluidic chip
temperature dependence on the resonant wavelength for
containing 1000 wells/channel was designed (Figure 16).
The principle highlighted in this study is paramount, as it silicon nitride. At a streptavidin concentration of 10−7 g/mL
shows the possibility to reduce the size of the device and keep the silicon nitride slot-ring resonator showed a 7× improve-
high binding efficiency. ment over the silicon ring resonator. Lastly, the researchers
Great importance must be attached to all these innovative used a silicon nitride slot-ring resonator to detect PSA, which
approaches, in terms of both applications and research is a known biomarker for prostate cancer. Instead of tagging
perspectives. Their combination and their development, thanks the SBP to biotin the researchers tagged SBP to anti-PSA to
to all the experimental tools available to date, will enable the immobilize the PSA antibody to the ring surface, achieving a
generation of innovative and smart platforms for detection or sensitivity of 10−8 g/mL.212
multiple targets. More recently, the group of Cognetti et al. demonstrated the
In terms of applications, silicon nitride ring resonators have use of integrated microring resonators on so-called “disposable
been used for biosensing with biological targets such as chips” embedding microfluidics for the detection of COVID-
biotin−streptavidin and prostate specific antigen (PSA) with
19213 and real-time sensing of lung epithelial inflammatory
promising results. This group utilized a silicon-binding protein
(SBP) which binds to silicon and silicon nitride and is used as markers (Figure 17).214 In these original works, the researchers
an anchoring molecule for other bioreceptors such as biotin.212 show the possibility to achieve organs-on-a-chip by measuring
The researchers conjugated the SBP to biotin and immobilized several targets secreted from cells maintained by a barrier. This
the protein on the ring resonator surface. Next, varying innovative concept opens the way to new types of applications
concentrations of streptavidin were exposed to the surface and and real-time multiplexing.
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■ COMBINED WGM SENSORS

Following the discussion of the recent state-of-the-art
precisely the role of point-of-care (POC) devices, which are a
general trend that consists of bringing sensors to the patient
biochemical detection techniques, we present in this section level (ambulatory practices). Recent technologies in portable
the interactions between WGMs and other technologies such diagnostics constitute the next wave of innovations in POC
as Raman, SERS, plasmonics, or acoustic wave measurements. sensing. Some current and emerging biosensors already enable
They benefit from both detection based on refractive index a transition between laboratory settings and home-based
changes and the other parameters induced by technological applications. The most prominent examples are blood glucose
association. monitoring and ecotoxicology, which have a constantly
The demonstration of the detection of single molecular or growing market already generating hundreds of billions of
cellular events has been essentially reported using gold or silver dollars each year. WGM sensors have the capability to detect
nanoparticles, structures, or nanofilms, enabling a plasmonic small RI changes, which makes them relevant for a wide range
enhancement. The generation of such enhancement can be of applications requiring the sensitive detection of molecular
designed through the high concentration of the electro- interactions. Some important developments are therefore
magnetic field supported by the resonant whispering-gallery focused on the end-user as the core of a multipurpose
plasmonic resonance. Using specific surface schemes or core− screening and information acquisition platform. This trend is
shell cavities, it is therefore possible to finely tune the radiative facilitated by progress in data processing with the aid of the
properties of light and generate appropriate SPR.215 This growing field of artificial intelligence (AI) and machine
phenomenon has been demonstrated theoretically and learning (ML) as well as the integration of miniaturized
experimentally under several configurations, often named sensors with smartphones and wearables (e.g., smart
“hybrid WGMs”. The main advantage of the plasmonic watches).228 To achieve such integration with very fragile
excitation is to significantly enhance the surface refractometric sensors (as is the case for WGM microstructures made of thin
sensitivity (typically within a range of 10−100×) and therefore silica walls), packaging is needed. Packaging provides the
enable the detection of smaller (or less frequent) events necessary protection to ensure long-term stability by enclosing
happening on the sensing area compared to the ones observed the microstructures in a protective material such as a polymer
on bare substrates.216−219 or a metal casing. Additionally, the packaging also helps to
In order to obtain a more specific sensor and to identify the maintain the integrity of both microfluidics and micro-
binding events more precisely through molecular fingerprints, resonators and preserve the quality of the WGM resonance
Raman scattering can be implemented on WGMs. The first signal. The use of packaging materials must not affect the
studies of stimulated Raman scattering (SRS) were performed efficiency of the coupling and, above all, the stability of the
on water and ethanol microdroplets. Following reports have light signal. Some with refractive indices that are too high
also shown the possibility to exploit SRS in aerosols and using compared to silica are therefore prohibited. With regard to
cavities (cavity-enhanced Raman scattering, CERS). It is packaging, this also implies sample delivery and the integration
important to mention that surface-enhanced Raman scattering of a microfluidic system to provide a continuous and
(SERS) is usually generated thanks to surface plasmon controlled supply of sample loading along the sensing area.
resonance and due to collective oscillation of electrons. In This allows the use of small volumes (typically starting from a
the case of WGMs, it is indeed possible to generate SERS using few μL) provided in small chips and capillary tubes with low
metallic nanoparticles or nanostructures220 as well as through energy consumption. Some devices even call on passive
nonplasmonic Raman scattering, produced from total internal microfluidics where the flow is performed through the capillary
reflection of the incident field.221 In this case, the Raman effect, while more advanced devices call on active microfluidics,
excitation beam overlaps with a WGM resonance, enabling its supported by micropumps and microvalves.229 The first
use for sensing.222−224 material of choice used to achieve this assembly is usually an
Alongside these detection techniques, imaging and local- elastomeric polymer, such as PDMS or hydrogels. Both inlets,
ization of nano/micrometric events located on the surface of outlets, diameters of the tubing, and flow channels need to be
the sensor or within the surrounding 3D matrix are essential determined. Then, a specifically designed wafer is used as a
objectives. This would make it possible to better understand reusable mold for the PDMS structure. This wafer is usually
binding mechanisms and also to locate cells and to made through photolithography, where the patterns are
discriminate their topography and their shape. One of the achieved by using a photosensitive polymer such as SU-8
methods to achieve this would be to implement photoacoustic that is selectively exposed to light. The surface is then
detection through WGM analysis. Opto-mechanics through immersed in successive etching solutions and thoroughly
surface acoustic wave WGMs has been demonstrated so far rinsed in order to clean the substrate from its upper layers and
and could lead to highly sensitive absolute thermometers, bring
leave the mold ready for the curing of PDMS.230 It is a well-
important applications in quantum technologies, and pave the
known material for research purposes and some commercial
way for miniaturized sensors for in situ bioimaging and
equipment. However, industries often prefer using other
biosensing.225−227 The combination of WGMs with different
polymers such as thermoplastics, which are cheaper at large
surfaces and the exploitation of different sensing technologies
scale and easier to handle using automated manufacturing
(combined-WGM) will therefore open new doors to achieve
breakthrough sensing modalities and bring interesting novelties tools. The absorption and hydrophobic effects provided by
to the field. PDMS channels also need to be handled, especially for their
long-term use inside equipment. When moving from an

■ CONCLUSION AND PERSPECTIVES

The demand for fast and reliable field-measurements, including
experimental research setup, the road often seems long to
reach total portability and sustainability. A second constraint,
in addition to the hardware, is also the signal analysis, which
biological and chemical parameters, is clearly evident. This is needs real-time communication and self-referencing.
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Although very promising, these sensors are very sensitive to Complete contact information is available at:
many external parameters such as temperature fluctuations, https://pubs.acs.org/10.1021/acssensors.2c02876
light polarization, or mechanical stress, among others,
requiring significant developments to overcome these practical Notes
limitations. Companies and spin-offs with commercial products The authors declare no competing financial interest.
are few but expanding, and recent years have shown increasing
interest in this technology. This momentum also initiates new
research purposes about their further integration and resolves
■ ACKNOWLEDGMENTS
We thank the support from the Belgian American Educational
all constraints in terms of reliability and reproducibility. Foundation (B.A.E.F.) https://baef.be providing the postdoc-
Prospects are therefore vast, and an impetus is clearly given to toral fellowship for Médéric Loyez. We acknowledge the
innovation and final applications. This widening of the vision support from the National Institutes of Health under the award
for “real products” including their integration and the number R21EB030845. The authors acknowledge the help of
association of various expertise within photonic laboratories Wenbo Mao, Di Jia, and Zhoutian Fu for their advice during
opens the way for several new challenges, slowly filling the gap the writing process.
between fundamental research, prototyping, and commercial-
ization. This also demonstrates the need for diversified profiles
and the combination of knowledge from different fields, as
■ VOCABULARY
Whispering gallery mode (WGM): a whispering gallery
highlighted in this review. The trends of future works are wave can be described as a wave traveling around a curved
therefore clearly oriented toward the needs for integrated surface by total internal reflection and constructively
imaging, multiplexing, and the study of new physical and interfering with itself, leading to enhanced light−matter
biochemical phenomena.231,232 It is important now to interactions for numerous applications such as lasing,
consolidate the experimental results obtained in research in nonlinear optics, and bio(chemical) sensing, among many
order to solidify their use on a larger scale. others.
In conclusion, this review portrays the contrast between a Optical resonator: an optical resonator can also be
family of photonic substrates (the fabrication of microscopic considered as a resonant cavity allowing light to circulate
WGM resonators) and surface functionalization chemistries in a closed path such as in a Fabry−Perot resonator. Many
(essentially using biological receptors). We summarized the structures are discussed in this review, such as microbubbles,
key steps to fabricate and achieve efficient biosensing tools microbottles, microrings and racetracks, microdroplets,
while providing adapted recipes, gathering the optical and microspheres, or microtoroids.
biochemical communities. WGMs have demonstrated excep- Biosensor: A biosensor is an analytical device measuring
tional sensitivities against a wide variety of molecular and specific analytes or biochemical reactions by generating
cellular targets so far. We outlined the strengths but also the signals proportional to the concentration of the target
weaknesses of the technology to guide future research on its analytes/reactions. They typically combine the use of a
improvement and innovation to find new avenues of specific sensing layer, which contains receptors that
application. The trends are currently in the biomedical field specifically bind to the target analyte, with a transducer
but are expanding to long-term monitoring such as for (detector).
environmental applications and quality biomonitoring. The Performance indicators: They are key parameters that
increasing support from industry indicates a high interest in the evaluate the value and progress of a sensor in specific
field and a constant need for new types of sensors with conditions. Examples include the limit of detection, quality
advanced features and performance. By presenting and factor, limit of quantification, sensitivity, and specificity,
analyzing a variety of projects and experimental results in among others.
this paper, we aim to provide an overview of the state-of-the- Optofluidics: Technological combination of optical sensors
art in the field, as well as to share emerging ideas that have the with microfluidics to bring samples directly in contact with
potential to drive cutting edge improvements in the near the area of interest, often optimizing both sample handling
future. and sensing performances.

■ AUTHOR INFORMATION
Corresponding Author
On-chip sensors: Integrated structures built on a chip,
providing a valuable platform for integration and miniatur-
ization for various applications.
Médéric Loyez − Department of Electrical & Systems
Engineering, Washington University, St. Louis, Missouri
63130, United States; orcid.org/0000-0001-8858-7413;
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