Biosensors: Design and Preparation of Sensing Surfaces For Capacitive Biodetection

biosensors
Review
Design and Preparation of Sensing Surfaces for Capacitive Biodetection
Perrine Robin and Sandrine Gerber-Lemaire *
Group for Functionalized Biomaterials, Institute of Chemical Sciences and Engineering, Ecole Polytechnique
Fédérale de Lausanne, 1015 Lausanne, Switzerland
* Correspondence: sandrine.gerber@epfl.ch
Abstract: Despite their high sensitivity and their suitability for miniaturization, biosensors are still
limited for clinical applications due to the lack of reproducibility and specificity of their detection
performance. The design and preparation of sensing surfaces are suspected to be a cause of these
limitations. Here, we first present an updated overview of the current state of use of capacitive
biosensors in a medical context. Then, we summarize the encountered strategies for the fabrication of
capacitive biosensing surfaces. Finally, we describe the characteristics which govern the performance
of the sensing surfaces, along with recent developments that were suggested to overcome their main
current limitations.
Keywords: non-faradaic impedance; capacitive detection; biosensors; electrochemical impedance

spectroscopy; surface functionalization; biomolecules immobilization; interdigitated electrodes
1. Introduction
Affinity-based biosensors operate by specifically capturing a biological target with
biological or synthetic capture agents such as aptamers, DNAzymes, single stranded DNAs
or antibodies. The binding event between the capture molecule and its target is later
translated into a readable signal.
Labelling the target molecules with fluorophores, magnetic beads, quantum dots or
enzymes, was reported to facilitate and amplify the readout signal. However, labelled-
detection methods are expensive and necessitate multi-step processes, hence are limited
for real-time detection. Label-free methods are therefore of interest for high throughput
biomolecules screening, portable devices and suitable for large-scale production [1].
Citation: Robin, P.; Gerber-Lemaire,
A variety of transducing methods have been described to translate the binding event
S. Design and Preparation of Sensing
between the targeted molecules and the probes (Figure 1). Conventional approaches include
Surfaces for Capacitive Biodetection.
optical, electrical or mass-sensitive techniques [2]. Electrical biosensors are great candidates
Biosensors 2023, 13, 17. https://
for miniaturized, portable and label-free detection, relying on potentiometric, voltammetric,
doi.org/10.3390/bios13010017
amperometric or impedimetric readout signals. Impedance-based sensors are themselves
Received: 23 November 2022 divided in faradaic or non-faradaic detection. Faradaic biosensors refer to the detection
Revised: 15 December 2022 of charge transfer across a membrane [3]. They often rely on electrochemical impedance
Accepted: 20 December 2022 spectroscopy (EIS) [4] which detects the binding events via the change of electron transfer
Published: 23 December 2022 resistance and double layer capacitance within a frequency range [5]. However, faradaic
detection is complex as it necessitates a wide window of frequencies [4]. It also requires
the addition of potentially hazardous redox couples, that can degrade biomolecules [4,6].
Copyright: © 2022 by the authors.
On the other hand, non-faradaic based sensors, also called capacitive sensors or third-
Licensee MDPI, Basel, Switzerland.
generation biosensors [7], detect the changes of capacitance at the electrode surface caused
This article is an open access article by the molecular binding events. These sensors have a high-sensitivity potential [4], and
distributed under the terms and do not require the addition of external reagent unlike other conventional methods, such
conditions of the Creative Commons as in situ hybridization or enzyme linked immunosorbent assays (ELISA) [4,8]. They offer
Attribution (CC BY) license (https:// a simple and rapid detection that can be inserted into portable devices [9]. Addition-
creativecommons.org/licenses/by/ ally, non-faradaic capacitive detection does not require trained laboratory personal or
4.0/). samples preparation, and is therefore interesting for point-of-care applications [4,10–12].
Biosensors 2023, 13, 17. https://doi.org/10.3390/bios13010017 https://www.mdpi.com/journal/biosensors

Biosensors 2023, 13, x FOR PEER REVIEW 2 of 22
Biosensors 2023, 13, 17 [9]. Additionally, non-faradaic capacitive detection does not require trained laboratory 2 of 22
personal or samples preparation, and is therefore interesting for point-of-care applications
[4,10–12]. Unfortunately, it has been reported that capacitive biodetection suffers from
poor to poor reproducibility
Unfortunately, [4,12–15]that
it has been reported andcapacitive
large standard deviation
biodetection [16,17],
suffers frompreventing
poor to poor
their translation for
reproducibility clinicaland
[4,12–15] application. Thesedeviation
large standard limitations havepreventing
[16,17], been suspected to arise
their translation
form
for sensing surface parameters
clinical application. such as itshave
These limitations cleanliness [4,18], homogeneity
been suspected to arise form [4] and insu-
sensing surface
lation [10,19–21].
parameters such as its cleanliness [4,18], homogeneity [4] and insulation [10,19–21].
Figure 1. Illustration of the different types of electrical sensors.

Figure 1. Illustration of the different types of electrical sensors.
Previous review articles focused on the different types of capacitive sensors [3,22,23],
Previous review articles focused on the different types of capacitive sensors [3,22,23],
geometries of electrodes [3], the use of nanomaterials for signal enhancement [5], the prepa-
geometries
ration of oftheelectrodes
electrodes[3],
bythe use of nanomaterials
molecular for signal
imprinting [1,24]. enhancement
A specific insight [5],
intothe prep-
capacitive
aration of the electrodes by molecular imprinting [1,24]. A specific insight into
immunosensors was also disclosed [25]. Here, we present the importance of the preparation capacitive
immunosensors was also
of sensing surfaces disclosed.
to overcome [25].
the Here, wewhich
limitations present the importance
capacitive of the
biosensors faceprepara-
in clinical
tion of sensing surfaces to overcome the limitations which capacitive biosensors
applications. Towards this goal, we will discuss the different functionalization strategies face in to
clinical applications. Towards this goal, we will discuss the different functionalization
immobilize the capture molecules on the electrodes, in addition to the influence of surface
strategies
propertiesto immobilize
(cleanliness the
andcapture molecules
homogeneity) onperformance
on the the electrodes,
andinreliability
addition oftothe
theresulting
influ-
ence of surface properties (cleanliness and homogeneity) on the performance
biosensors. Additionally, methods to obtain well-insulated surfaces and efforts made toand reliabil-
ityavoid
of thenon-specific
resulting biosensors.
binding of Additionally, methods
the target molecules to obtain
at the surfacewell-insulated
will be examined.surfaces
and efforts made to avoid non-specific binding of the target molecules at the surface will
be 2. Methodologies for Capacitive Biosensor Detection
examined.
Two main electrode geometries were reported for capacitive sensing, leading to two
2. Methodologies for methodologies
distinct capacitive Capacitive Biosensor
for theDetection
detection of binding events between capture
molecules
Two main and their targets.
electrode The were
geometries most reported
commonfor is based on potentiostatic
capacitive capacitance
sensing, leading to two
measured
distinct at themethodologies
capacitive electrode/solution interface.
for the detectionInofthis case, the
binding capture
events molecules
between captureare
immobilized
molecules on thetargets.
and their working electrodes.
The As an alternative,
most common is based oninterdigitated
potentiostaticelectrodes
capacitancehave
received growing attention over the last three decades for capacitive detection
measured at the electrode/solution interface. In this case, the capture molecules are im- [3]. In this
case, theon
mobilized recognition
the workingelements are immobilized
electrodes. on a substrate
As an alternative, in between
interdigitated the electrodes,
electrodes have
which undergo capacitance changes upon binding to the molecular targets [22].
received growing attention over the last three decades for capacitive detection [3]. In this
case, the recognition elements are immobilized on a substrate in between the electrodes,
Biosensors 2023, 13, 17
which undergo capacitance changes upon binding to the molecular targets [22]. 3 of 22
2.1. Potentiostatic Capacitance

Potentiostatic capacitance measurements at the electrode/solution interface is based
2.1. Potentiostatic Capacitance
on the theory of the electrical double-layer. The electrodes are generally made of a con-
ductivePotentiostatic
metal, oftencapacitance
covered by measurements at the
an insulating layer onelectrode/solution
which the capture interface is based
biomolecules are
on the theory of the electrical double-layer. The electrodes are generally made
immobilized. When the electrode is immersed in an electrolyte solution, at a given poten-of a con-
ductive metal, often covered by an insulating layer on which the capture biomolecules are
tial, the surface charge (qm) and solution charge (qs) equilibrate according to qm = −qs.
immobilized. When the electrode is immersed in an electrolyte solution, at a given potential,
Charged species and dipoles contained in the solution will orientate towards the surface,
the surface charge (qm ) and solution charge (qs ) equilibrate according to qm = −qs . Charged
forming the double-layer. It was demonstrated experimentally that the system can be rep-
species and dipoles contained in the solution will orientate towards the surface, forming
resented as three capacitors in series, as illustrated in Figure 2. The insulating layer repre-
the double-layer. It was demonstrated experimentally that the system can be represented as
sents the first capacitor, Cins. The second capacitor, Crec, corresponds to the immobilized
three capacitors in series, as illustrated in Figure 2. The insulating layer represents the first
molecules and their target, in addition to the double layer. The last capacitor (Cd) repre-
capacitor, Cins . The second capacitor, Crec , corresponds to the immobilized molecules and
sents the diffuse layer. Overall, the capacitance of the system Ctot is given by Equation (1)
their target, in addition to the double layer. The last capacitor (Cd ) represents the diffuse
[22].
layer. Overall, the capacitance of the system Ctot is given by Equation (1) [22].
1
𝐶𝑡𝑜𝑡 = 11
Ctot = 1 + 1 +1 1
1 (1)
(1)
C
𝐶 +
𝑖𝑛𝑠 C
𝐶 +
𝑟𝑒𝑐 C
𝐶𝑑
ins rec d
Figure 2. Schematic representation of electrode–solution interfaces for capacitive detection of

biomolecules. Crec increases after capture of the target biomolecules.
Figure 2. Schematic representation of electrode–solution interfaces for capacitive detection of bio-
molecules.
WhenCrec increases
the after capture
target molecule of thetotarget
binds biomolecules.
the capture probe, it results in a change of Crec
that can be detected via the monitoring of the total capacitance, Ctot . As shown in the
When(1),
Equation theCtarget molecule binds
tot is dominated to the
by the capture
smallest probe, it results
capacitance of theinthree.
a change of Crec that
Therefore, if
can be detected via the monitoring of the total capacitance, C . As
present, the insulating layer should be thin with a high dielectric constant to result
tot shown in the Equationin a
(1),
highCC tot is value.
ins dominated by the smallest capacitance of the three. Therefore, if present, the
insulating layer should be thin with a high dielectric constant to result in a high Cins value.
2.2. Interdigitated Electrodes
2.2. Interdigitated Electrodes
Capacitive sensors based on interdigitated electrodes (IDE) are also called interdigital
capacitive
Capacitivesensors. Thebased
sensors use ofonthese electrodes electrodes
interdigitated results in enhanced
(IDE) are also impedance changes,
called interdigi-
higher
tal signal-to-noise
capacitive ratio,
sensors. The and
use increased
of these speed results
electrodes of detection [4].
in enhanced impedance changes,
higher The geometry ofratio,
signal-to-noise IDEsandis illustrated in Figure
increased speed 3. Usually,
of detection [4]. IDEs are fabricated by
lithography
The geometry of IDEs is illustrated in Figure 3. Usually, The
techniques on glass substrates or silicon wafers. IDEselectrodes widthbyand
are fabricated li-
spacing can vary from tenths of nanometers to tenths of microns [3].
thography techniques on glass substrates or silicon wafers. The electrodes width and spac- In this configuration,
the can
ing sensor surface
vary from consists
tenths ofofnanometers
two paralleltometal
tenths electrodes
of micronsseparated by aconfiguration,
[3]. In this dielectric surface.
the
The capture probe can be immobilized on the electrodes, on
sensor surface consists of two parallel metal electrodes separated by a dielectric the insulating surface,
surface. in
between,
The capture orprobe
on bothcan[19]. The capacitance
be immobilized on theof the electrodes
electrodes, on theisinsulating
linked tosurface,
the dielectric
in be-
constant of the surface in between the electrodes with the Equation (2)
tween, or on both [19]. The capacitance of the electrodes is linked to the dielectric constant[22].
of the surface in between the electrodes with the Equation (2) [22].
ε × ε0 × A
C= (2)
𝜀 × d𝜀0 × 𝐴
C= (2)
where ε stands for the dielectric constant 𝑑of the substrate between the plates,
ε0 = 0.8.85419 pF/m (vacuum permittivity), A is the area of the electrodes and d the
distance between them.
Thus, when molecule binds to the surface, it causes a change in the dielectric proper-
ties of the substrate, that can be retrieved by monitoring the changes of capacitance of the
electrodes.
Instead of measuring a capacitance change, an alternative approach consists of meas-
uring the changes of distances between the capacitor plates. These devices are composed
Biosensors 2023, 13, 17 4 of 22
of a rigid electrode and a flexible one, often a membrane. Capacitive membranes were
previously reported by Tsouti et al. [3] and therefore are not further described.
Figure 3. Graphical representation of interdigitated electrodes on a surface (left) and cross section
Figure 3. Graphical representation of interdigitated electrodes on a surface (left) and cross section
view of an interdigitated biosensor after probe capture (right). Adapted from Tsouti et al., [3].
view of an interdigitated biosensor after probe capture (right). Adapted from Tsouti et al. [3].
3. Current
Thus, State
whenof Capacitive
molecule Biosensors
binds Used it
to the surface, incauses
Clinical Applications
a change in the dielectric prop-
ertiesDue to substrate,
of the the varietythat
of can
capture probes which
be retrieved can be immobilized
by monitoring the changes on the electrodes
of capacitance of
surface,
the capacitive biosensors can be designed for a wide range of medical applications.
electrodes.
Viruses, unicellular
Instead and disease
of measuring markerschange,
a capacitance were detected in biological
an alternative fluids,
approach via non-fara-
consists of mea-
suring the changes ofThe
daic measurements. distances between
following the capacitor
sections focus on plates. These
the most devices
recent arewhich
studies composed
ena-
of a rigid
bled electrode and
to significantly a flexible
decrease one, often
the limits a membrane.
of detection Capacitive
(LoD) compared to membranes were
previous systems
previously reported
and/or addressed by Tsoutidetection
capacitive et al. [3] and therefore
in complex are not further
biological described.
samples.
3. Current
3.1. State of Capacitive Biosensors Used in Clinical Applications
Infections
Due to the variety
Immunosensors of capture
have probesfor
been reported which can be immobilized
the capacitive detection of onviruses
the electrodes
such as
surface, capacitive biosensors can be designed for a wide range of medical
Influenza virus [26,27], foot and mouth disease [28], Hepatitis B [29], Norovirus applications.
[30], Zika
Viruses,
virus [31]unicellular and disease
and SARS-CoV-2 markers
[32]. weregenosensors
However, detected in biological
were more fluids,
widelyviadescribed
non-faradaic
for
measurements. The following sections focus on the most recent studies
viral capacitive detection, and generally displayed lower LoD than immunosensors. which enabled toIn
significantly
1999, Bergreen decrease the limits
et al. could detectofCytomegalovirus
detection (LoD) compared
with a limittoofprevious
detectionsystems and/or
of 0.2 aM from
addressed
DNA standard capacitive detection
fragments [13].inMore
complex biological
recently, samples.
commercial DNA sequence of West Nile
virus
3.1. could be detected with a limit of detection (LoD) of 1.5 aM [4].
Infections
While these studies investigated the specificity of the obtained sensors with non-tar-
Immunosensors have been reported for the capacitive detection of viruses such as
geted genes, none of them reported the detection of viral targets in complex biological
Influenza virus [26,27], foot and mouth disease [28], Hepatitis B [29], Norovirus [30], Zika
samples. Matrix effect, coming from the interaction of non-targeted biomolecules with the
virus [31] and SARS-CoV-2 [32]. However, genosensors were more widely described
sensors surface, can have deleterious effect on the sensing efficiency. For example, in 2017,
for viral capacitive detection, and generally displayed lower LoD than immunosensors.
Cheng et al. reported a genosensor for the detection of Herpes 1 virus [33]. While a LoD
In 1999, Bergreen et al. could detect Cytomegalovirus with a limit of detection of 0.2 aM
of 10.7 aM could be achieved in standard buffer, the limit increased to 0.21 fM in neat
from DNA standard fragments [13]. More recently, commercial DNA sequence of West
serum, likely due to unwanted interactions of non-targeted biomolecules with the sensor
Nile virus could be detected with a limit of detection (LoD) of 1.5 aM [4].
surface [33]. To our knowledge, no capacitive sensors displaying attomolar detection has
While these studies investigated the specificity of the obtained sensors with non-
been reported to detect viruses in real biological samples.
targeted genes, none of them reported the detection of viral targets in complex biological
Diagnosing
samples. a viralcoming
Matrix effect, infectionfromcanthealso be performed
interaction by recognizing
of non-targeted antibodies,
biomolecules with i.e.,
the
performing serological tests. In that regard, Zeng et al. recently reported an impedimetric
sensors surface, can have deleterious effect on the sensing efficiency. For example, in 2017,
biosensor
Cheng that
et al. can detect
reported SARS-CoV-2
a genosensor forantibodies (Abs).
the detection of The authors
Herpes studied
1 virus [33]. two enhance-
While a LoD
of 10.7 aM could be achieved in standard buffer, the limit increased to 0.21 fMprobing
ment techniques (illustrated in Figure 4) to improve the LoD of the system, (1) by in neat
the targeted
serum, likelyAbsduewith a gold nanoparticle
to unwanted interactions(AuNP)-tagged
of non-targetedsecondary antibody
biomolecules andsensor
with the (2) by
using dielectric
surface electrophoresis
[33]. To our knowledge, no (DEP). In the sensors
capacitive case of displaying
the AuNPsattomolar
enhancement strategy,
detection has
been reported to detect viruses in real biological samples.
Diagnosing a viral infection can also be performed by recognizing antibodies,
i.e., performing serological tests. In that regard, Zeng et al. recently reported an im-
pedimetric biosensor that can detect SARS-CoV-2 antibodies (Abs). The authors studied
two enhancement techniques (illustrated in Figure 4) to improve the LoD of the system,
(1) by probing the targeted Abs with a gold nanoparticle (AuNP)-tagged secondary anti-
body and (2) by using dielectric electrophoresis (DEP). In the case of the AuNPs enhance-
ment strategy, the presence of the nanoparticles enhances the measurable signal, that can
reduce the LoD value. In the case of DEP enhancement, targeted biomolecules can be
selectively moved and concentrated to the sensing surface due to the dielectric properties
Biosensors 2023, 13, x FOR PEER REVIEW 5 of 22
Biosensors 2023, 13, 17 5 of 22

the presence of the nanoparticles enhances the measurable signal, that can reduce the LoD
value. In the case of DEP enhancement, targeted biomolecules can be selectively moved
and concentrated to the sensing surface due to the dielectric properties of each molecule.
of each molecule. While a LoD of 2 µg/mL was obtained with the DEP enhancement
While a LoD of 2 μg/mL was obtained with the DEP enhancement technique, the authors
technique, the authors could detect down to 200 ng/mL with AuNPs [34].
could detect down to 200 ng/mL with AuNPs [34].
Figure4.4.Enhancement
Figure EnhancementofofSARS-CoV-2
SARS-CoV-2antibodies
antibodiesdetection
detectionvia
viathe
theuse
useofofAuNPs
AuNPs(left)
(left)orordielec-
dielec-
trophoresis force (right). Adapted from Zeng et al. 2022 [34].
trophoresis force (right). Adapted from Zeng et al. 2022 [34].
Non-faradaicimpedimetric
Non-faradaic impedimetric sensors
sensors werewere also
also reported
reported for for
otherother
typetype of pathogens,
of pathogens, in-
including protozoan, worms and bacteria. In 2022, Figueroa-Miranda
cluding protozoan, worms and bacteria. In 2022, Figueroa-Miranda et al. reported a aptasen-et al. reported a ap-
tasensor based on a graphene surface for the detection of a malaria marker,
sor based on a graphene surface for the detection of a malaria marker, Plasmodium falciparum Plasmodium
falciparum
lactate lactate dehydrogenase,
dehydrogenase, with a limit with a limit of detection
of detection 0.78 fM inof 0.78 fM
diluted in diluted
human serumhuman
[35],
serum [35],
allowing the allowing
detection the detection of parasitemia,
of low-density low-density and parasitemia,
surpassing andthe
surpassing the LoD
LoD previously
previously
achieved via achieved via Faradaic
Faradaic detection detection [36–38].
[36–38].
Worm’santigens
Worm’s antigenswere
werealso
alsodetected
detectedvia vianon-faradaic
non-faradaicimpedance
impedancemeasurements.
measurements.
Zhouetetal.,
Zhou al.,reported
reportedthetheuse
useofofgold
goldelectrodes
electrodesmodified
modifiedwith withSchistosoma
Schistosomajaponicum
japonicumanti-
anti-
bodies
bodiestotodetect
detectthetheworms’
worms’antigens.
antigens.AALoD LoDofof0.10.1ng/mL
ng/mLwas wasreached
reachedininPBS.,
PBS.,and
andthethe
selectivity
selectivityofofthethesensor
sensorwas
wasassessed
assessedbybycomparing
comparingthe thecapacitance
capacitancechanges
changeswith withother
other
proteins
proteins[39].
[39].
The
Thepresence
presenceofofbacteria
bacteriacan
caneither
eitherbebedetected
detectedvia viathe
thepresence
presenceofoftoxins,
toxins,orordirectly
directly
detecting
detectingthe theunicellular
unicellularorganisms.
organisms. While capacitive
capacitive detection
detection of oftoxins
toxinswaswasreported
reportedin
infood
food[40,41]
[40,41]ororininwater
water [42],
[42], detection
detection of of toxins
toxins hashasnotnot
beenbeen reported
reported for for diagnosis
diagnosis pur-
purposes. On the contrary, direct detection of bacteria has been reported
poses. On the contrary, direct detection of bacteria has been reported and is described and is describedin
inthe
theUnicellular
Unicellulardetection
detectionsection.
section.
3.2.
3.2.Cancer,
Cancer,Chronic
Chronicand
andInflammatory
InflammatoryDiseases
Diseases
Early detection and diagnosis of cancer,
Early detection and diagnosis of cancer, chronic oror
chronic inflammatory diseases
inflammatory cancan
diseases drasti-
dras-
cally improve the chances of survival [43–45]. Capacitive detection of such biomarkers has
tically improve the chances of survival [43–45]. Capacitive detection of such biomarkers
been used in this sense for a variety of pathologies.
has been used in this sense for a variety of pathologies.
A variety of cancer biomarkers were selected as targeted molecules for early cancer
A variety of cancer biomarkers were selected as targeted molecules for early cancer
detection. Recent studies pointed to the detection of markers with a sufficient LoD to
detection. Recent studies pointed to the detection of markers with a sufficient LoD to en-
enable cancer early diagnosis. For example, in 2018, Arya et al. [46] reported a LoD of
able cancer early diagnosis. For example, in 2018, Arya et al. [46] reported a LoD of 0.1
0.1 ng/mL in non-diluted serum. As patients suffering from breast cancer possess around
ng/mL in non-diluted serum. As patients suffering from breast cancer possess around 14
14 to 75 ng/mL of Her2 markers in their blood, this aptasensor could be used in the future
to 75 ng/mL of Her2 markers in their blood, this aptasensor could be used in the future
for diagnosis [46].
for diagnosis [46].
The detection of marker for chronic and inflammatory diseases was also reported via
capacitance measurements. For example, the capacitive detection of C-reactive protein
(CRP), a biomarker for cardiovascular disease risks, sepsis and other tissue inflammation
was extensively studied [11,12,47–49]. A decade later, Macwan et al. [48], could surpass the
Biosensors 2023, 13, 17 6 of 22
sensitivity of previous CRP electrochemical sensors [11,12] and reached a 10 fg/mL LoD
in both PBS buffer and serum by switching to an interdigitated sensing device integrated
sputtered with nanofibers to enhance the sensitivity and selectivity of the sensing surface.
Human chitinase-3-like protein 1, a marker for tissue inflammation and cardiac disease
was also detected by capacitive systems with a LoD of 0.07 µg/L. This is 300 times lower
than ELISA method, currently used for this marker detection [50]. However, this study was
performed in diluted serum to prevent the matrix effect and complementary studies should
thus be performed to assess the LoD in real biological sample. Other biomarkers have
been targeted for chronic-disease diagnosis, such as LDL-cholesterol [6], interleukin-3 [51]
or transferrin [20,52]. Nampt, an obesity marker for which Park et al. improved the
binding affinity with the surface compared to previously reported techniques, could be
detected in the nanomolar range [53]. Finally, Kumar Sharam et al. recently reported
the detection of amyloid beta within 5 s with a LoD of 0.1 fg/mL for the diagnostic of
Alzheimer disease [54].
3.3. Unicellular Organisms

Capacitive sensors for whole cell detection and analysis were developed over the last
decade. The first occurrence of unicellular organism detection by non-faradaic measure-
ments dates back 2004, for E. Coli detection in food samples by using an immunosensor
targeting antigens present at the bacteria surface [55]. Since then, the detection of Salmonella
(bacteria), Cryptosporidium (protozoan) in food or water samples was disclosed. In 2013,
Couniot et al. also reported a simulation for optimal design of capacitive sensors for bacte-
ria detection [56]. Recently, Borsel-Oliu et al. reported the use of a 3D IDE platform that
evaluates the response of bacteria to antibiotics [57]. This platform could in the future be
used for a variety of toxicity evaluations.
Non-faradaic biosensors have also been developed for eukaryotic cell detection.
In 2022, Zhang et al. [58], detailed the detection of peripheral blood mononuclear cell
(PBMCs), that can indicate the immune function state of a patient. While PMBCs cells
are normally found in concentrations ranging from 0.7 to 6.2·106 cells/mL, their sensor
displayed a LoD of 104 PBMCs/mL, with the possibility to quantify the cells. However,
further experiments would be needed to assess the LoD in real samples for potential
clinical applications.
Finally, yeast detection has also been reported with a non-pathogenic strain,
Saccharomyces cerevisiae. A LoD value of 0.1 ng/mL was achieved, with a detection range of
0.4 to 18 ng/mL [59]. We expect that further developments could lead to the detection of
pathogenic strains for clinical use.
Viruses, unicellular organisms and diseases markers detected via capacitive biosensing
are summarized in Table 1.
Biosensors 2023, 13, 17 7 of 22
Table 1. Non-faradaic impedimetric biosensors reported for clinical applications. Capacitive sensors for food and waste control were not included.
Target Sensor Type Electrode Material Sensor Preparation Detection Range LoD Ref.
SAM formation of
Foot and Mouth disease Immunosensor Gold N/D N/D [28]
thiol-modified epitope
Parylene coating, followed by <100 pg/mL in both buffer
Hepatitis B Immunosensor Gold nanoislands 0.1–1000 ng/mL [29]
glutaraldehyde cross-linking and serum
Magnetic nanobeads coated 2
1.6.10 ELD50 /mL of
Influenza H5N2 Immunosensor Gold 1.5·101 –1.5·105 ELD50/ml [27]
with antibodies purified virus
Antibody immobilization through
Influenza H5N1 Immunosensor Gold 101 –105 EID50/mL 103 EID50 /mL in buffer [26]
adsorbed Protein A
Polyaniline followed by
Norovirus Immunosensor Gold streptavidin coupling and 1 fg/mL–1 ng/mL 60 ag/mL in buffer [30]
biotinylated Ab immobilization
Polyvinyl alcohol, Alignate EDC/NHS coupling of antibodies 6.6 × 103 PFU/mL
Zika virus Immunosensor N/D [31]
and Polyaniline on alginate in buffer
Poly(3,4-
147 TCID50 /mL of virus
Viral infection Immunosensor ethylenedioxythiophene) Antibodies adsorption N/D [32]
from culture fluid
SARS-CoV-2 virus polystyrene sulfonate
APTES modification, followed by
Genosensor Platinium/Titanium 5 µM–10 nM 10 nM in PBS [60]
phosphoramidite linkage
200 ng/mL and 2 µg/mL
in buffer
APTES modification, followed by
SARS-CoV-2 antibodies Immunosensor Gold N/D (for AuNP and DEP [34]
EDC/NHS coupling of antibodies
enhancements,
respectively)
SAM formation of 0.2 aM of pure fragment
Cytomegalovirus Genosensor Gold N/D [13]
thiolated oligonucleotides in buffer
SAM formation of
West Nile virus Genosensor Gold 3–33 aM 1.5 aM in buffer [4]
thiolated oligonucleotides
SAM formation of 10.7 aM in buffer
Herpes 1 virus Genosensor Aluminum 0.2 fM–0.2 pM in serum [33]
thiolated oligonucleotides 0.21 fM in neat serum
SAM formation of
Papillomavirus Genosensor Indium oxide 0.1 pM–0.1 µM 20 fM in buffer [61]
thiolated oligonucleotides
Graphene modified aptasensors
0.78 fM in diluted human
Aptasensor Graphene oxide linked to grapehene surface via 0.78 fM–100 nM [35]
serum
π- π stacking
Malaria enzyme marker 0.77 fM in 50%
Pathogen markers Thiolated-aptamers
human serum
Aptasensor Gold Polyethylene glycol layer added 1 pM–100 nM [62]
1.49 pM in whole
to reduce non-specific adsorption
human serum
Schistosoma japonicum Antibody immobilization through
Immunosensor Gold 0.4–18 ng/mL 0.1 ng/mL in PBS [39]
antigen adsorbed Protein A
Biosensors 2023, 13, 17 8 of 22
Table 1. Cont.
Parylene coating, followed by
SSAT Immunosensor Gold/Titanium 1.25–10 mg/L 1.25 mg/mL in buffer [9]
glutaraldehyde cross-linking
SAM formation of 0.1 ng/mL in
Aptasensor Gold 1 pM–100 nM [46]
thiol-modified aptamers non-diluted serum
Her2
SAM formation of
0.2 ng/mL in
Aptasensor Gold mercaptopropionic acid followed 0.2–2 ng/mL [8]
diluted serum
by peptide coupling
SAM formation of
Her4 Affimer-based sensor Gold 1 pM–100 nM 1 pM in non-diluted serum [63]
Cancer biomarkers cysteine-modified aptamers
Carboxylic-modified gold
Protein-affinity-based nanoparticles layer formed via 10 pM pure antigens
PMSA Aluminum electrodes 10 pM–100 nM [64]
sensor thiol-gold bond, followed by in buffer
peptide coupling
Platelet derived APTES modification followed by
Aptasensor Silicium 1–50 µg/mL 1 µg/mL in buffer [65]
growth factor phosphoramidite bond
Carboxylic acids introduced via
Squamous
Immunosensor Gold SAM formation, followed by N/D 2.43 µg/mL in buffer [14]
carcinoma antigen
peptide coupling
Immunosensor Gold SAM formation, followed by 25 pg/mL–25 ng/ml 25 pg/mL in PBS [11]
peptide coupling
SAM formation of
Aptasensor Gold 200 pg/mL–2 ng/mL 200 pg/mL in PBS [12]
thiol-modified aptamers
ZnO thin film deposition,
0.10 µg/mL in human
Protein C reactive Immunosensor Gold followed by succinimidyl 0.01–20 µg/mL. [47]
serum and whole blood
propionate crosslinking
Chronic or
inflammatory diseases Carbon fibers sputtered on the
electrode, followed by 10 fg/mL in PBS and
Immunosensor Gold 1 fg/mL–1 ng/mL [48]
dithiobis(succinimidyl) serum buffer
propionate cross-linking
SAM formation of carboxylic 1 ng/mL of purified
Immunosensor Nickel 1–250 ng/ml [66]
acids, followed by antigens
Immobilization of streptavidin via
SAM formation, followed by
Myeloperoxidase Immunosensor Gold 1 pg/mL to 1 µg/ml ~1 pg/mL in buffer [49]
biotinylated-antibodies
conjugation
Biosensors 2023, 13, 17 9 of 22
Table 1. Cont.
Gold nanoparticles spread on the
Immunosensor Screen printed electrode electrode, followed by adsorption 0.1–12.5 0.2 ng/mL in buffer [67]
of the antibody
Troponin
Amino groups introduced via 0.01–5 ng/mL in PBS 0.01 ng/mL in PBS
Immunosensor Alumiium SAM formation, followed by buffer 0.07–6.83 ng/mL in 0.07 ng/mL in [68]
cross-linking with glutaraldehyde human blood serum human serum
Thiourea introduction via SAM
Human
Immunosensor Gold formation, followed by 0.1 µg/L–1 mg/L 0.07 µg/L in buffer [50]
chitinase-3-like protein 1
glutaraldehyde cross-liking
Silicon doped Introduction of amine followed by
Immunosensor N/D N/D [20]
semiconductor glutaraldehyde cross-linking
Chronic or Transferrin Electropolymerization of
inflammatory diseases
Immunosensor Glass carbon phenylenediamine, followed by 0.1–45.0 ng/mL in 0.061 ng/mL in PBS [52]
glutaraldehyde cross-coupling
Amine introduction followed by
Interleukin-3 Immunosensor Zeolite-Iron 3–100 pg/mL 3 pg/mL in buffer [51]
peptide coupling
Interleukin-6 Immunosensor Gold SAM formation, followed by 25 pg/mL–25 ng/ml 25 pg/mL in PBS [11]
peptide coupling
Nampt Aptasensor Gold SAM thiol aptamers 1–50 ng/mL 1 ng/mL in diluted serum [53]
Amine introduction followed by
Amyloid beta Aptasensor Platinium/Titanium 0.001–10 µM 1 fg/mL in buffer [54]
peptide coupling
Amino groups introduced via
120 pg/mL of
LDL-Cholesterol Immunosensor Gold SAM formation, followed by N/D [6]
pure antigens
cross-linking with glutaraldehyde
Carboxylic groups introduced via
Peripheral blood
Immunosensor Gold SAM formation followed by N/D 104 cells/mL in buffer [34]
mononuclear cell
peptide coupling
Unicellular organisms Carboxylic acids introduced via

44 cells/mL in
CD34+ T-cells Immunosensor Gold SAM formation, followed by 50–1 × 105 cells/mL [69]
diluted serum
peptide coupling
Bacteria answers Polyethyleneimine layer for
/ Tantalum silicide N/D N/D [57]
to antibiotics bacteria adsorption (non-specific)
Abbreviations: N/D: not detailed. TCID50 : tissue culture infective dose. ELD50 : 50% Egg Lethal Dose. EID50 : 50% Egg Infective Dose. APTES: aminopropyltriethoxy silane.
SAM: Self-assembled monolayer.
Biosensors 2023, 13, 17 10 of 22
4. Capacitive Sensing Surfaces Preparation, and Limitations

4.1. Capacitive Sensors Limitations
Capacitance biosensors, whether they are based on IDEs or potentiostatic capacitance
measurements, necessitate the immobilization of biomolecules on a surface. In the case
of IDEs, the molecules are immobilized on the interface between the electrodes. For
potentiostatic capacitance measurements, probes are immobilized directly on the electrode,
eventually covered with an insulating layer (see in Figures 2 and 3). The capture molecules
can be antibodies, to further detect an antigen or a pathogen (immunosensor), single-strand
DNAs, for the detection of RNA or DNA single strands (genosensor) or aptamers, for the
fabrication of aptasensors.
Unfortunately, even after years of progress in capacitive biodetection, challenges re-
main. Poor reproducibility [4,12,12–15] and large standard-deviation [16,17] of capacitive
biosensors have been reported and linked to non-optimal parameters of their sensing
surfaces [4,13,19]. Critical parameters in the capacitive sensing surfaces preparation have
been raised, such as the probe immobilization strategy [13,19,52], surface cleanliness [4,18],
homogeneity [4] and insulation [10,19–21]. Obtaining a high specificity with capacitive
sensors is also challenging as any adsorbed biomolecules at the sensing surface can pre-
vent target binding or generate a false-positive signal [10,16,17,62]. After describing the
most common strategies for the preparation of sensing surfaces, the surface parameters
suspected to affect capacitive sensors behavior are presented, along with the solutions
recently reported to overcome these limitations.
4.2. Biomolecules Immobilization Techniques

Various immobilization strategies have been reported for the immobilization of capture
molecules on capacitive sensing surfaces. The functionalization procedures may depend
on the type of electrodes used and the nature of capture molecules immobilized. Selection
of the functionalization pathway for a capacitive sensor is crucial, as it can affect drastically
its performance [23].
While electrodes are generally made from metals such as gold [4,8,11,12,14,19,21,26–
29,33,39,40,46–48,50,53,55,58,62,70–79], platinum [41], graphene [35,80], glass carbon [7,52]
or aluminum [33,64,68], electrodes made of titanium [9,60,63,81,82], nickel [66], or sili-
cium [65,83–85] are less described even though they are great candidates for capacitive
measurements. Indeed, they display high insulation properties, can present smooth sur-
faces with homogeneous functionalities for capture probe immobilization [23]. Finally, few
more exotic materials have been reported as electrodes material. For example, tantalum
was selected for antibodies [86] or bacteria detection [57]. Recently, polymers were reported
for the production of electrodes for capacitive biodetection. Park et al. described the
use of electrodes made of a conductive polymer layer of PEDOT:PSS for the detection of
SARS-CoV-2 [32]. Frias et al. used polyvinyl alcohol, alginate and polyaniline to fabricate
electrodes for Zika virus detection [31].
Gold electrodes are generally favored for capacitive measurements as they are fre-
quently encountered in other detection transduction techniques such as surface plasmon
resonance, quartz crystal microbalance or reflection absorption IR spectroscopy, thus allow-
ing for dual-mode detection and direct comparison of readout signals [18].
Two main strategies are commonly described for the immobilization of capture
molecules on sensing surfaces. A first method consists of the formation of a self-assembled
monolayer (SAM) at the surface of metallic electrodes. This layer can be directly made of
the capture molecule, or of a linker that is later used to conjugate the capture molecule [87].
A second methods relies on the deposition a thin insulating film at the surface of the
electrodes, generally made of a polymer or silanes, followed by the conjugation of the
capture molecule to this first layer [23].
The most frequently reported strategies for the immobilization of capture probes on
the electrode and/or insulating layer are detailed in the following sections.
Biosensors 2023, 13, 17 11 of 22
4.2.1. Self-Assembled Monolayer (SAM) Formation on Metal Electrodes

The adsorption of SAMs of biomolecules on metals and metal oxides is frequently
used for the modification of electrodes. Due to the strong affinity of sulfur atoms for
gold, the most reported SAM technique is based on the incubation of gold surfaces with
thiol-modified biomolecules [87].
Thiol-modified single-strand DNA (ssDNA) probes were immobilized on gold elec-
trodes for cytomegalovirus detection [13]. ssDNA immobilization on aluminum electrodes
was also performed for the detection of West Nile [4] and Herpes viruses [33], but also on
indium oxide for the detection of papillomavirus [61]. The immobilization of thiol-modified
aptamers on gold electrodes was described for the detection of obesity markers [53], can-
cer [46] or inflammatory diseases markers [12], but also thrombin [21], and malaria [62].
Finally, other capture molecules such as affirmers [63], peptide-nucleic acids [74], and
epitopes [28], were exploited for diagnosis application.
Another strategy consists of adding a first layer of alkyl-thiol chain on the electrode,
to which the capture molecules is further conjugated. This strategy was reported for
the immobilization of antibodies [6,11,14,27,48,50,58,69,71,72,75,77–79], ssDNA [13] and
aptamers [8,80]. Numnuam et al. also reported the immobilization of histones on gold
electrodes for the detection of DNA traces [88]. Figure 5 illustrates the differences between
direct surface functionalization with thiol-containing biomolecules via SAM formation,
Biosensors 2023, 13, x FOR PEER REVIEW 11 and
of 22
the use of a thiol linker for covalent conjugation of the capture molecule.
Figure5.5.Comparison
Figure Comparisonofofdirect
directSAM
SAMformation
formationofofthiol-containing
thiol-containingbiomolecules
biomolecules(left)
(left)and
anduse
useofofaa
thiol linker (right) for the immobilization of biomolecules at the surface of a metallic electrode.
thiol linker (right) for the immobilization of biomolecules at the surface of a metallic electrode.
Thedirect
The directimmobilization
immobilizationofofbiomolecules
biomoleculesvia viaSAM
SAMformation
formationisisthe thesimplest
simplestway waytoto
functionalize metallic surfaces with capture probes [89]. However,
functionalize metallic surfaces with capture probes [89]. However, it requires the use it requires the useofof
thiol-containing biomolecules or the modification of native biomolecules
thiol-containing biomolecules or the modification of native biomolecules with a thiol group with a thiol
group beforehand. Procedures involving a linker were shown to provide
beforehand. Procedures involving a linker were shown to provide more stable function- more stable func-
tionalized
alized surfaces.
surfaces. Bergreen
Bergreen et al. et al. compared
compared theimmobilization
the two two immobilization strategies
strategies [13].one
[13]. On On
one hand,
hand, a 26 bases
a 26 bases thiol-modified
thiol-modified ssDNA ssDNA was directly
was directly immobilized
immobilized on goldonelectrodes,
gold electrodes,
while
while
on on the
the other other
hand, anhand,
8-basean 8-base
probe wasprobe
coupledwasviacoupled via carbodiimide
carbodiimide chemistry tochemistry
a cysteamine to a
cysteamine monolayer previously deposited on the gold electrode. The
monolayer previously deposited on the gold electrode. The pre-functionalization strategy pre-functionaliza-
tion astrategy
with with a linker
linker resulted resulted
in enhanced in enhanced
selectivity selectivity
of the of the sensor,
sensor, despite the usedespite
a shorterthe use a
probe.
shorter
The probe.
length Thelinker
of the lengthisofalso
theof linker is also of importance.
importance. Mirsky et al.Mirsky
studied et al. studied
various various
SAMs of
SAMs of mercapto-alkyl
mercapto-alkyl derivativesderivatives
deposited on deposited on goldfollowed
gold electrodes, electrodes, followed bywith
by conjugation conjuga-
Abs.
tionstudy
The withconcluded
Abs. The that
study concluded
longer that longer
mercapto-alkyl mercapto-alkyl
layers were preferable, layers
duewere preferable,
to spontaneous
due to spontaneous
desorption of shorterdesorption of shorter
chains. Finally, chains.
the use Finally,
of linkers thealso
has useimpact
of linkers on has
the also impact
insulating
properties of sensing surfaces which will be discussed in Section 4.5.
on the insulating properties of sensing surfaces which will be discussed in Section 4.5
4.2.2.
4.2.2.Covalent
CovalentImmobilization
Immobilizationononan
anInsulating
InsulatingLayer
Layer
Beside
Beside SAM formation on metallic electrodes,capture
SAM formation on metallic electrodes, molecules
capture moleculescan also
can bebe
also immobi-
immo-
lized
bilized on a surface-deposited insulating layer presenting reactive functionalitiesfurther
on a surface-deposited insulating layer presenting reactive functionalities for for fur-
chemical conjugation.
ther chemical conjugation.
The most common routes for deposition of the insulating layer include silanization
methodologies [19,20,41,60,65,81,82,86] and polymerization strategies. Electropolymeri-
zation of tyramine [70,76], phenylenediamine [52], and polyaniline [83,85] were reported
for the functionalization of surfaces with amine or carboxylic-containing polymers. Ther-
Biosensors 2023, 13, 17 12 of 22
The most common routes for deposition of the insulating layer include silanization
methodologies [19,20,41,60,65,81,82,86] and polymerization strategies. Electropolymeriza-
tion of tyramine [70,76], phenylenediamine [52], and polyaniline [83,85] were reported for
the functionalization of surfaces with amine or carboxylic-containing polymers. Thermal
deposition of parylene was also proposed by Jung et al. for the fabrication of a capac-
itive biosensor [29]. The thickness of the insulating layer is also critical to keep a high
sensitivity [29,83]. Using platinum electrodes covered by tin oxide films, Choudhury et al.
demonstrated that film thickness below 100 nm resulted in good insulation properties and
higher sensitivity than thicker films for the capacitive detection of immunoglobulin [83].
Functionalization of surfaces with polymers instead of silanes was found to improve
the sensitivity and specificity of the resulting device, which might be due to surface
insulation enhancement [9]. Deposition of a polymeric layer can also be performed with a
simpler fabrication procedure than silanization [85]. However, if the sensor is inserted in a
microfluidic platform, we believe that the adhesion of the polymer to the surface must be
carefully controlled to avoid the removal of the polymer due to the pressure.
Various chemical linkages can be then used to immobilize capture molecules on the
formed insulating layer. Among them, peptide bond coupling has been extensively re-
ported. It has the advantages of being stable under a wide range of pH [90], compatible with
many types of surfaces and suitable for the conjugation of a variety of biomolecules due to
the presence of amines and/or carboxylic groups in their structure. Carbodiimide chem-
istry was described for the covalent conjugation of Abs to silanized surfaces [19,86]. The
use of cross-linkers was also widely reported. Glutaraldehyde is the most common spacer
for the immobilization of biomolecules and was highlighted for the conjugation of Abs
to parylene [29], poly(o-phenylenediamine) [52], polytyramine-[70], polyaniline [83,85],
and amino-silane modified surfaces [20,81,82]. Similar strategy was reported for the im-
mobilization of phages to a polytyramine insulating layer for the capacitive detection of
Salmonella spp. Noticeably, the sensor could be used up to 40 times following alkaline
treatment to regenerate the active sensing surface [76]. Other cross-linkers such as N-γ-
maleimidobutyryloxy succinimide ester [41,55] or dithiobis (succinimidyl propionate) [47]
were disclosed for the conjugation of Abs to capacitive sensing surfaces.
The different methods for covalently conjugating biomolecules to insulating layers are
now summarized in Table 2.
As alternative chemical linkage to peptide-bond formation, a few other types of chem-
ical conjugations were reported in the context of the fabrication of capacitive biosensors.
When considering nucleic acids as capture molecules, phospharamidite bond formation
is of interest for their covalent immobilization. Liao and Cui reported the attachment of
a phosphated aptamer with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as
coupling agent to an APTES-modified surface [65]. Finally, Varlan et al. reported the
silanization of TiO2 surfaces with glycidoxysilane for the immobilization of antibody via
epoxide ring-opening for hormone detection [81]. Figueroa-Miranda et al. described the
modification of a graphene oxide surface with pyrene-modified aptamers via π stacking.
The resulting sensing surface was later used for malaria detection [35]. EDC/NHS coupling
was reported by Yagati et al. for the covalent attachment of aptamers on pyrenebutyric
acid previously immobilized on a graphene IDEs via π-π stacking. The obtained aptasen-
sors were studied for the detection of thrombin in blood [80]. The use of biotin/avidin
complex formation was also disclosed for the immobilization of antibodies on gold sur-
faces, addressing the capacitive detection of cardiovascular protein biomarkers [49] and
norovirus [30].
are propionate)
now are now
N--maleimidobutyryloxy
summarized
The different [47]
N--maleimidobutyryloxywere
summarized
in Table disclosed
incovalently
2. Table
succinimide for2. theester
succinimide conjugation
ester
[41,55] ofbiomolecules
[41,55]
or Abs orto
dithiobis capacitive
dithiobis
(succinimidylsensing
(succinimidyl surfaces.
propio- propio-
are
kaline
nate) The
kaline now
simpler
[47]
with
layers.
summarized
kaline
different
treatment
treatmentR:fabrication
were
many The tomethods
treatment
methods
disclosed
types
Capture
infor
toTable
regenerate
todifferent
regenerate for
of procedure
methods
surfaces
molecule. thefor
the 2.
regenerate
thecovalently
than
for
conjugation
and the
active
active sensing conjugating
active
conjugating
sensing
silanization
covalently
suitable of sensing
surface
surface
Abs
for the [85].
to surface
biomolecules
[76].
[76].
conjugating
capacitive
conjugation Other
However, [76].
Other to
Other
ifatothe
ofcross-linkers
biomolecules
sensing insulating
insulating
sensorcross-linkers
cross-linkers
to
surfaces.
variety layers
such
is layers
such as assuch
inserted
of insulating
biomolecules in as
layers
are nate)
are
now now nate)
[47] were[47]
summarized were
disclosed
summarized
The different in disclosed
in
Table
methods for
Table 2. the
2. for
succinimide
for the
conjugationconjugation
succinimide
covalently ester of Abs
ester
[41,55]
conjugating ofto Abs
[41,55]
or to
capacitive capacitive
or
dithiobis
biomolecules sensing
dithiobis sensing
surfaces. surfaces.
(succinimidyl
(succinimidyl
to insulating propio-
layers propio-
adue
The microfluidic
different
are
to now
the platform,
methods
summarized ofsuccinimide
for we believe
incovalently
Table for2. ester
that [41,55]
the
conjugatingadhesionor dithiobis
of the
biomolecules (succinimidyl
polymer
to to the
insulating propio-
surface
layers must
Table
Table The
Table
2. Methods
2. Methods 2.presence
The different
different
Methods
reported
reportedmethods
for
amines
methods
reported
thethe
for for
for
covalent
and/or
covalently
the
covalent
carboxylic
covalently
covalent
conjugation conjugating
conjugation
conjugation
groups
ofconjugating
capture
Captureof of
capture
in their
biomolecules
capture
biomolecules
structure.
biomolecules toon
biomolecules
biomolecules on to Carbodiimide
insulating
insulating
insulating on
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insulating
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Function Introduced arenate)
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removal
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capacitive
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due
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Agentcovalent theCapture
conjugation
Moleculesensor couldof Abs toLinkage
be Chemical
Chemical used silanized
up to 40Reference
Linkagesurfaces
times
Reference [19,86].
following
Reference
Biosensors 2023, 13, x FOR PEER REVIEW Molecule Molecule [86] [86] 12[86]
of 23
The use of cross-linkers
alkaline treatment toCoupling was
regenerate also widely
the active reported. Glutaraldehyde
Capture
sensing surface [76].
Capture
Capture Other is the most[86]
cross-linkers common such
Function
Function Function
Introduced
Introduced Introduced Coupling Agent Agent
N-Cyclohexyl-N′-(2-
Coupling Agent Chemical
Chemical Chemical
Linkage
Linkage
Peptide bond Linkage Reference
Reference Reference
spacer for the immobilization ofsuccinimide
biomolecules Antibody
and was highlighted for the [86] [86]
conjugation
Function Introduced as N-γ-maleimidobutyryloxy
Coupling Agent
N-Cyclohexyl-N′-(2-morpho-
Molecule
Capture
Molecule Molecule
ester
Molecule [41,55] or dithiobis
Chemical
Peptide
Linkage (succinimidyl [86] of
Reference
Abs
morpholinoethyl)carbodiimide
to parylene-[29], poly(o-phenylenediamine)- Antibody
Antibody Antibody Peptide
[52], Peptide bond bond bond polyaniline-
polytyramine-[70],
propionate) [47] were disclosed
linoethyl)carbodiimide
N-Cyclohexyl-N′-(2-morpho- for the conjugation of Abs to capacitive sensing
[86] [86] surfaces.
[86]
3-Triethoxysilylpropylamine
3-Triethoxysilylpropylamine [83,85],N-Cyclohexyl-N′-(2-morpho-
insulation N-Cyclohexyl-N′-(2-morpho-
andenhancement
amino-silane modified
[9]. Deposition
Antibody
Antibody
surfaces [20,81,82].
of a polymericAntibody
Peptide
Peptide
Similar
layer bond
can
bond
strategy
Peptide
also be bondwas
performed
[86]reported with for a
N-Cyclohexyl-N′-(2-morpho- [86]
3-Triethoxysilylpropylamine the
simpler immobilization
Thefabrication linoethyl)carbodiimide
different methods of
procedure phages
forthan to a
covalently Antibody
polytyramine
silanizationconjugating
Antibody [85]. Peptide
insulating
Peptide
However, bond
biomolecules bond
layer for the
to insulating
if the sensor [86]capacitive
is inserted layers in
3-Triethoxysilylpropylamine Antibody Peptide bond [86] [86]
3-Triethoxysilylpropylamine detection
are
a now
microfluidic of Salmonella
summarized platform, spp.
Table
we Noticeably,
in
N-Cyclohexyl-N′-(2-morpho- 2.
believe that the
the
Antibodysensor
adhesion could of be
Peptide
the used
bond
polymer up toto40 times
the following
surface
[86] [86] [86]
must
3-Triethoxysilylpropylamine N-Cyclohexyl-N′-(2- AntibodyPeptide Peptide
bond bond
activeAntibody
0
alkaline
be carefully linoethyl)carbodiimide
N-Cyclohexyl-N
treatment
controlled to-(2-avoid the
to regenerate theremoval Antibody
Antibody
sensing surfacePeptide
of the polymer duePeptide
[76]. bond
thebond
Other
to cross-linkers such
pressure.
Biosensors3-Triethoxysilylpropylamine linoethyl)carbodiimide
2023, 13, x FOR PEER REVIEW N-Cyclohexyl-N′-(2-morpho- N-Cyclohexyl-N′-(2-morpho- 13 of 23
3-Triethoxysilylpropylamine 2. N-Cyclohexyl-N′-(2-morpho-
as N-γ-maleimidobutyryloxy
3-Triethoxysilylpropylamine Table Methods
Various reported
chemical linkages succinimide
for thecan covalent
be then Antibody
conjugation
Antibody used Antibody
ester to of Peptide
[41,55]capture
Peptide
immobilize orPeptide
bondbond bond[19]
dithiobis
biomolecules
capture (succinimidyl
moleculeson [19]the
[19]insulating
on
3-Triethoxysilylpropylamine layers.
formed R: insulating
propionate) Capture[47] molecule.
were layer.disclosedAmong for them,
the conjugation
peptide bond of Abscoupling
to capacitive has beensensing
[19][19] surfaces.
extensively
3-Triethoxysilylpropylamine Peptide [19]
Antibody
Antibody Antibody Peptide
Peptide bond bond bond
3-Triethoxysilylpropylamine reported. It has the advantages
EDC/NHS of being stable under
Capture a wide range of pH [90],
[19] compatible
[19]
Function Introduced with The EDC/NHS
EDC/NHS Peptide bond [19]
3-Triethoxysilylpropylamine many types Coupling
different ofmethods
surfaces Agent
for
andcovalently
suitable Antibody
Antibody
for Aptamer
conjugating
the conjugation
Antibody
Molecule
Chemical
Peptide bond
biomolecules
of a Linkage
Peptidevarietybond of[19]Reference
to insulating
biomolecules
[19]
layers
[19]
EDC/NHS
EDC/NHS [19] [19]
3-Triethoxysilylpropylamine are
3-Triethoxysilylpropylamine due now
to thesummarized
presence of inamines
Table 2.
EDC/NHS and/or Antibody
carboxylic
Antibody groups Peptide
Peptide
Antibody Peptidein their
bond bond
structure.
Peptide bond Carbodiimide
3-Triethoxysilylpropylamine Antibody bond
Peptide bond
chemistry wasEDC/NHS EDC/NHS
EDC/NHS
described for the covalent
Antibody
conjugation of Abs to silanized surfaces
3-Triethoxysilylpropylamine EDC/NHS EDC/NHS Antibody Peptide bond [19] [19,86].
[19]
3-Triethoxysilylpropylamine Table 2. ofMethods reportedwas for also
the covalent Antibody
conjugationAntibody of Peptide
capture Peptide
bond bond[19]on insulating
biomolecules
The use EDC/NHS
cross-linkers widely reported. Glutaraldehyde is the most common
3-Triethoxysilylpropylamine layers. R: Capture molecule. EDC/NHS EDC/NHS
3-Triethoxysilylpropylamine spacer for the immobilization of biomolecules Antibody
Antibody andAntibody
was Peptide
highlighted
Peptide Peptide
bondbondforbond the[65] [65] [65] of
conjugation
EDC/NHS
EDC/NHS EDC/NHS
3-Triethoxysilylpropylamine Abs to parylene-[29],
N-Cyclohexyl-N′-(2-morpho-poly(o-phenylenediamine)- Capture[52], polytyramine-[70], [65]polyaniline-
[65][19]
[65]
Function Introduced
3-Triethoxysilylpropylamine Coupling Agent
linoethyl)carbodiimide Chemical
Peptide
Peptide Peptide
bond bond Linkagebond Reference
[83,85],N-Cyclohexyl-N′-(2-morpho-
and amino-silane
N-Cyclohexyl-N′-(2-morpho- modified surfaces Aptamer [20,81,82].
MoleculeAptamer Similar strategy was reported
[86] [65]for
N-Cyclohexyl-N′-(2-morpho- Aptamer Antibody Peptide Peptide
Peptide bond bond [65] [65] [65]
3-Triethoxysilylpropylamine the immobilization EDC/NHS
of 0 -(2-
linoethyl)carbodiimide phages to a polytyramine
N-Cyclohexyl-N′-(2- Aptamer insulating bond layerbond
Peptide for the
bond capacitive
N-Cyclohexyl-N
N-Cyclohexyl-N′-(2-morpho- Aptamer Aptamer Peptide
Antibody bond
Peptide [65]
Peptide bond [65] [65]
2023, 13, x FOR PEER detection
Biosensors
of Salmonella
morpholinoethyl)carbodiimide spp. Noticeably, theAptamer
REVIEWN-Cyclohexyl-N′-(2-morpho-
linoethyl)carbodiimide Aptamer
sensor could
Aptamer Peptidebe used
bond
Peptideup tobond40 times following
[65] 12 of 22
alkaline treatment N-Cyclohexyl-N′-(2-morpho-
to regenerate the active sensing surface
N-Cyclohexyl-N′-(2-morpho- Aptamer
3-Triethoxysilylpropylamine Peptide[76].bond Other cross-linkers [65] [65] such
3-Triethoxysilylpropylamine N-Cyclohexyl-N′-(2-
Aptamer Peptide
Peptide Peptide
bond bond bond[65]
3-Triethoxysilylpropylamine as N-γ-maleimidobutyryloxy
succinimide AptameresterAptamer[41,55] or dithiobis (succinimidyl
[60]
3-Triethoxysilylpropylamine propionate) [47] were disclosed for the conjugation
linoethyl)carbodiimide Peptide
ofPeptide
Abs Peptide
tobondbond bond
capacitive sensing
[60][60]surfaces.[60]
N-Cyclohexyl-N′-(2- Aptamer
Aptamer Aptamer
The use of cross-linkers was also widely reported. Glutaraldehyde Peptide bond is the most [60]
[86] common
3-Triethoxysilylpropylamine N-Cyclohexyl-N′-(2-morpho-
morpholinoethyl)carbodiimide ssDNA [60][60] [60]
Biosensors 2023, 13, x FOR PEER REVIEW spacer for the immobilization of biomolecules
ssDNA and
ssDNA was
Peptide highlighted
Peptide Peptide
bond for
bond the
bond to insulating layers conjugation
13 of 23 of
The different methods
N-Cyclohexyl-N 0 -(2- for covalently
linoethyl)carbodiimide conjugating
ssDNA
Antibody biomolecules
Peptide bond
Peptide bond [60] [60] [65]
Biosensors 2023, 13, x FOR PEER REVIEW N-Cyclohexyl-N′-(2-morpho-
Abs morpholinoethyl)carbodiimide
tomorpholinoethyl)carbodiimide
parylene-[29], poly(o‐phenylenediamine)- [52], polytyramine-[70], 13 of 22
polyaniline-
[60]
3-Triethoxysilylpropylamine are now summarized in Table
linoethyl)carbodiimide 2. ssDNA
ssDNAssDNA Peptide
Peptide bondbond
3-Triethoxysilylpropylamine linoethyl)carbodiimide
linoethyl)carbodiimide ssDNA Peptide Peptide bond bond [60]
[83,85], andN-Cyclohexyl-N′-(2-morpho-
amino-silane modified surfaces [20,81,82]. Similar strategy was reported for
3-Triethoxysilylpropylamine morpholinoethyl)carbodiimide
linoethyl)carbodiimide ssDNAssDNAssDNA Peptide
Peptide bond bond bond [60] [60]
Peptide
3-Triethoxysilylpropylamine the linoethyl)carbodiimide
immobilization
Table 2. Methods of phages
linoethyl)carbodiimide to a polytyramine insulating
reported for the covalent conjugation of capture biomolecules on insulating layer for the capacitive detec-
N-Cyclohexyl-N′-(2-morpho- ssDNA Peptide bond [60][60]following[60]
3-Triethoxysilylpropylamine tion
layers. R:of linoethyl)carbodiimide
Salmonella
Capture molecule. spp. Noticeably, the sensorssDNA couldPeptide
be used bondup tobond
Peptide 40 times al-
3-Triethoxysilylpropylamine, linoethyl)carbodiimide
linoethyl)carbodiimide ssDNA [19]
kaline treatment to regenerate the active sensing surface [76]. Other cross-linkers
Biosensors 2023,
Biosensors 13,
Biosensorsx FOR
13,2023,PEER REVIEW
13,PEER
x FORREVIEW
Aminobutyldimethylmethoxysilane
3-Triethoxysilylpropylamine PEER REVIEW
2023,3-Triethoxysilylpropylamine
x FOR ssDNA Peptide Peptide
bond bond 13 13 of 2213 of as
of 22 such 22
linoethyl)carbodiimide ssDNA
linoethyl)carbodiimide ssDNA
Capture
Antibody Peptide bond
Peptide bond
Function Introduced N--maleimidobutyryloxy EDC/NHS
Coupling Agentsuccinimide ester [41,55]Chemical or dithiobis Linkage [20] [20] [20]
(succinimidyl
Reference [20]
propio-
3-Triethoxysilylpropylamine Molecule [20]
nate) [47] were disclosed
Glutaraldehyde Glutaraldehyde
Glutaraldehyde
Glutaraldehyde for the conjugation Antibody
Antibody
Antibody of Abs
Antibody to capacitive
Peptide
Peptide Peptide
bond bond bond [20][70]
Peptide sensing
bond surfaces.
[20]
[20]
Glutaraldehyde Peptide
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde Antibody Peptide
Antibody
Antibody Peptide bondbond
bond [20] [20]
Aminobutyldimethylmethoxysilane Peptide bond [20]
3-Triethoxysilylpropylamine,
Aminobutyldimethylmethoxysilane Antibody Peptide bond
Glutaraldehyde Antibody
Polytyramine
Glutaraldehyde
for covalently conjugating biomolecules to[20]
insulating layers
3-Triethoxysilylpropylamine, Glutaraldehyde
Antibody Peptide
Peptide bond bond bond
3-Triethoxysilylpropylamine, are now summarized in Table 2. Antibody Peptide [20][82] [20]
3-Triethoxysilylpropylamine, Glutaraldehyde [19]
3-Triethoxysilylpropylamine, Glutaraldehyde
Glutaraldehyde Antibody Peptide bond
Aminobutyldimethylmethoxysilane Glutaraldehyde AntibodyAntibody Peptide
Peptide bondbondbond[20][20] [20]
Peptide
3-Triethoxysilylpropylamine, Avidin
3-Triethoxysilylpropylamine,Table 2. Methods EDC/NHS
reported for the covalent conjugation of capture biomolecules on insulating lay-
3-Triethoxysilylpropylamine Glutaraldehyde
Glutaraldehyde
Glutaraldehyde Antibody AntibodyPeptidePeptide
bond bond[82][82][86]
[82]
Biosensors 2023, 13, x FOR PEER REVIEW
3-Triethoxysilylpropylamine, ers. R: Capture molecule. Antibody 13
[76]of 22
3-Triethoxysilylpropylamine, N-Cyclohexyl-N′-(2-
Glutaraldehyde
Glutaraldehyde Peptide bond
Glutaraldehyde
Glutaraldehyde AntibodyPeptide
Avidin
Avidin
bond
Peptide
Peptide bond
Peptide
Peptide
bond bond
bond
Glutaraldehyde
morpholinoethyl)carbodiimide Avidin Avidin Peptide
Capture bond
Function Introduced
3-Triethoxysilylpropylamine Coupling Agent Phage Chemical Linkage Reference
3-Triethoxysilylpropylamine Molecule
Polytyramine
3-Triethoxysilylpropylamine [82]
3-Triethoxysilylpropylamine Avidin
3-Triethoxysilylpropylamine [65]
[70][85] [70]
N-Cyclohexyl-N′-(2- Peptide bond [70] [86]
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde
morpholinoethyl)carbodiimide Antibody
Antibody
Antibody Peptide
Peptide Peptide bond [70][70]
bond
bond [70]
Glutaraldehyde
N-Cyclohexyl-N′-(2-morpho- Antibody Peptide bond
Polyaniline
Polytyramine Glutaraldehyde
Glutaraldehyde
Glutaraldehyde Antibody Antibody
Peptide Peptide
bond bond
Polytyramine AntibodyAntibody PeptidePeptide
bond bond
Polytyramine
Polytyramine
Polytyramine [19]
Polytyramine [83]
Antibody Peptide bond [76]
EDC/NHS [70]
Glutaraldehyde
Glutaraldehyde Peptide Peptide
bond bond [19]
Polyaniline PhageAntibody
Polytyramine
3-Triethoxysilylpropylamine Antibody Peptide bond[76][76] [76]
EDC/NHS
Glutaraldehyde
PeptidePeptide
bond bond
Polytyramine Phage
Phage Phage
[85] [76]
Polytyramine [65]
Polytyramine
Glutaraldehyde
Peptide bond
bond
N-Cyclohexyl-N′-(2- Antibody Peptide
Antibody bond
3-Triethoxysilylpropylamine, Phage Peptide bond
Polyaniline [81]
Polytyramine The use of cross-linkers was also widely reported. Glutaraldehyde is the most common
Polytyramine
Polytyramine N--maleimidobutyryloxy succinimide ester [41,55] or dithiobis (succinimidyl propio-
Biosensors 2023, 13, x FOR PEER REVIEW spacer for the immobilization of biomolecules and was highlighted for the conjugation 13 of 22 of
nate) [47] were disclosed for the conjugation of Abs to capacitive sensing surfaces.
Abs to parylene-[29], poly(o‐phenylenediamine)- [52], polytyramine-[70], polyaniline-
[70]
[70]13 of 22
Polytyramine
[83,85], and amino-silane modified surfaces [20,81,82].Peptide
Similarbond
strategy was 12 offor
reported 22
Biosensors 2023, 13, x FOR PEER REVIEW Glutaraldehyde
Glutaraldehyde Antibody [76]
Biosensors 2023, 13, x FOR PEER REVIEW The different methods for covalently conjugating
Antibody biomolecules
Peptide bond of 22 13layers
to insulating
13 of 22
the immobilization of phages to a polytyramine insulating layer for the capacitive [76]
[70]
[76] detec-
Biosensors 2023, 13, 17
are now summarized in Table 2. Peptide bond 14 of 22
tion of Salmonella spp. Noticeably, the Phage
Glutaraldehyde sensor couldPeptide
be used up to 40 times following al-
Polytyramine
Phage Peptide bond
bond [76] common
kaline
The usetreatment
of to regenerate
cross-linkers was the widely
also active sensing
reported.
Phage surface [76]. Other is
Glutaraldehyde cross-linkers
theonmost such as
Polytyramine Table 2. Methods reported for the covalent conjugation of capture biomolecules insulating lay-
Polytyramine
Polytyramine N--maleimidobutyryloxy
spacer for the immobilization
Glutaraldehyde succinimide
of ester
biomolecules [41,55]
and was or dithiobis
highlighted
Peptide bond (succinimidyl
for the propio-
conjugation
[70] of
Biosensors 2023, 13, x FOR PEER REVIEW ers. R: Capture
Table 2. Cont. molecule. 13 of 22
nate)to
Abs [47] were disclosed
parylene-[29], for the conjugationPhage
poly(o‐phenylenediamine)- of Abs to capacitive
[52], sensing
polytyramine-[70], surfaces.
polyaniline-
Glutaraldehyde Antibody Peptide bond [70]
[76]
[83,85], and amino-silane modified surfaces Capture
[20,81,82]. Similar strategy was reported for
[85] Reference
Biosensors
Biosensors 2023,
Function
Function
2023, Introduced
Introduced
13, xREVIEW
13, Polytyramine
x FOR PEER FOR PEER REVIEW Coupling Coupling
Agent
Glutaraldehyde Agent Capture Molecule
AntibodyMolecule
Chemical
Chemical
Peptide bond [70]13 of[76]
Linkage
Linkage [70]
Reference
22 12 of 22
The different
the immobilization methods
of phagesfortocovalently
a conjugating
polytyramine
Phage biomolecules
insulating layer for to
the [85]
insulating
capacitive
[85] layers
detec-
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde AntibodyAntibody Peptide bond
Peptide bond
Peptide bond [76]
Polytyramine are now Glutaraldehyde
summarized Table 2. Antibody
inNoticeably, Peptide bond
Polytyramine
Polyaniline
tion of Salmonella spp.
Glutaraldehyde
Glutaraldehyde the sensor
Phage
Antibody could be used
Peptide
Peptide bondup to 40 times following al-
bond
Polytyramine Polytyramine
Polyaniline kaline treatment to regenerate the activePhage sensing surface [76]. Other cross-linkers such as
Polyaniline The use of cross-linkers was also widely reported. Glutaraldehyde is theonmost common
Polytyramine Table 2. Methods reported forsuccinimide
N--maleimidobutyryloxy the covalent conjugation
ester [41,55]of capture biomolecules
or dithiobis (succinimidylinsulating lay-
propio-
[70][76][76] [76]
Biosensors 2023, 13, x FOR PEER REVIEW spacer
ers. R: for the
Capture immobilization
molecule.
nate) [47] Glutaraldehyde
were of biomolecules and was highlighted
disclosed for the conjugation of Abs to capacitive sensing [85] for the
13 conjugation
of 22
surfaces.[86] of
Biosensors 2023,Polytyramine Glutaraldehyde
Glutaraldehyde
13, x FOR PEER REVIEW Abs to parylene-[29],Glutaraldehyde Antibody
poly(o‐phenylenediamine)- Peptide
Peptide
[52], bond
bond
polytyramine-[70],
Peptide bond
Peptide bond [76] 14 of 23
[83] polyaniline-
N-Cyclohexyl-N′-(2-morpho- AntibodyPhage
Phage
Phage CapturePeptidePeptide
Function
Polytyramine
Polytyramine Introduced [83,85], andGlutaraldehyde
Glutaraldehyde
amino-silane
Coupling modified
Glutaraldehyde Agent surfaces Antibody
[20,81,82]. bond
Similar
Chemical
Peptide
Peptide bond bond[70]
bondstrategy [76]
Linkage [83]
was [76]
reported
[83]Reference for
Polytyramine The different methods for
linoethyl)carbodiimidecovalently conjugating
Antibody
Phage Molecule biomolecules to insulating
[85] layers
Polyaniline
3-Triethoxysilylpropylamine the immobilization of
Glutaraldehyde phages
Glutaraldehyde
Glutaraldehyde to a polytyramine
Antibody insulating
Peptide layer
bond
Peptide for the
bond [85]
capacitive detec-
Polyaniline
are now summarized in Table 2. Antibody
AntibodyPhage Peptide bond
Peptide bond
Biosensors 2023, 13, x FOR PEER REVIEWtion of Salmonella
Polyaniline Glutaraldehyde
spp. Noticeably,
Glutaraldehyde Phage
theAntibody
sensor couldPeptide
be used bond
up to 40 times following
14 of 23 al-
Polyaniline
Polytyramine Antibody Peptide bond
Polytyramine
Polyaniline
Polytyramine kaline2.treatment
Table to regenerate
Methods reported for thethe activeconjugation
covalent sensing surface [76].biomolecules
of capture Other cross-linkers
on such as
[76][85]insulating lay-
Biosensors 2023, 13, x FOR PEER REVIEW N--maleimidobutyryloxy
ers. R: Capture molecule. succinimide ester [41,55] or dithiobis (succinimidyl [70]13 [85]
[83]of propio-
22 [85]
Glutaraldehyde Antibody Peptide bond sensing [85] [86]
Biosensors 2023,Polyaniline
13, x FOR PEER REVIEW nate) [47] Glutaraldehyde
were disclosed for the conjugation
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde Antibodyof Abs to capacitive
Peptide bond surfaces.[19]
14 of 23
Glutaraldehyde Antibody Phage Peptide
Antibody
Antibody bond
Peptide
Peptide bond bond
Peptide
CapturePeptide bond bond
Polyaniline
Biosensors 2023, 13, x FOR PEER REVIEW Glutaraldehyde Antibody [85] [85]12 of 22
Function
Polytyramine
Polyaniline Introduced
Polyaniline
Polytyramine
Coupling Agent
MoleculePeptide
Chemical
Peptide
bond bond[76][83]Reference
Linkage
Polyaniline Glutaraldehyde Antibody Peptide bondto insulating layers
TheGlutaraldehyde
different
Glutaraldehyde methods for covalently
Glutaraldehyde Antibody conjugating
Antibody
Peptide
Peptide
Peptide
biomolecules
bond
bondbond
EDC/NHS Antibody
Polyaniline Polyaniline
3-Triethoxysilylpropylamine, are now summarized in
Glutaraldehyde Table 2. Phage [81]
Polyaniline
Polyaniline
3-Triethoxysilylpropylamine, Antibody Peptide bond [85]
Polytyramine
Biosensors 2023, 13, x FOR PEER REVIEW [81]
[81]
[83] 14 of 23
Aminobutyldimethylmethoxysilane The use of cross-linkers Glutaraldehyde was also widelyAntibodyreported. Glutaraldehyde is [85]
the most common
Table 2. Methods Peptide
reported for the covalent conjugation of capture biomolecules bond on insulating
spacer for the Glutaraldehyde
immobilization of biomolecules Antibodyand was highlighted
Peptide bond for[70]
the[83]
[76] [83] [83] lay-
conjugation of
3-Triethoxysilylpropylamine, ers. R: Capture molecule.
Glutaraldehyde Antibody Peptide bond 13 of 22[86]
Polyaniline Abs to parylene-[29],
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde
None
Glutaraldehyde poly(o‐phenylenediamine)-
Antibody
Antibody [52],
Peptide polytyramine-[70],
bond
Peptide
Peptide bond
Antibody Peptide
Antibody bondbond
polyaniline-
[81] [19]
Polyaniline
Aminobutyldimethylmethoxysilane Glutaraldehyde Antibody [85]
Biosensors 2023, 13, x FOR PEER REVIEW andN-Cyclohexyl-N′-(2-morpho-
[83,85], GlutaraldehydeNone
amino-silane Phage
modified surfaces Epoxide
[20,81,82].
Capture ring-open-
Similar
Peptidestrategy
bond was [65]12 offor
reported 22
Polyaniline
Polyaniline
Polyaniline
Polytyramine
Function Introduced Glutaraldehyde
None
Coupling Agent Antibody Antibody
Antibody
Antibody Peptide Peptide
bond
Chemical bond
Linkage Reference
Glutaraldehyde Epoxide
Epoxidebond
Molecule ring-open-
ing
ring-open- [81]
the immobilization of phages to a polytyramine
N-Cyclohexyl-N′-(2-morpho- Antibody Peptide
insulating layer
Peptide for the capacitive detec-
Glycidoxypropyldimethylethoxysilane
Polyaniline
Polyaniline Glutaraldehyde Antibody
AntibodyAntibody Peptideing bond bond
Peptide bond
[83]
Polyaniline
Polyaniline tion of Salmonella EDC/NHS
spp. Noticeably, the sensor
linoethyl)carbodiimide could be useding upbond
to 40 times [81]following al-
Glutaraldehyde Aptamer
Antibody
Antibody Peptidebond
Peptide
Aminobutyldimethylmethoxysilane [83][81]
Aminobutyldimethylmethoxysilane kaline
The usetreatment to regenerate
of cross-linkers was also the widely
active sensing
reported.surface [76]. Other is
Glutaraldehyde cross-linkers
the most common such as
spacer for the immobilization
Glutaraldehyde succinimide
of ester
biomolecules [41,55]
and was or dithiobis
highlighted for [85]
(succinimidyl
[76]
the conjugationpropio- of
Glutaraldehyde
None
None Antibody Peptide
Antibody Peptide bondbond [70][55]
Polyaniline nate) [47] were disclosed
Glutaraldehyde
Abs to parylene-[29],
Glutaraldehyde for the conjugation
poly(o‐phenylenediamine)-
Antibody of Epoxide
Abs to
[52],
Peptide ring-open-
capacitive
bond sensing
polytyramine-[70], surfaces.[86]
polyaniline-
Polyaniline Glutaraldehyde Antibody Peptide
Peptide bond
Epoxidebondring- [83][55] [55][83]
[81] [81][19]
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde
N-y-maleimidobutyryloxy Antibody
succini- Antibody
Phage
Antibody
Antibody Peptide
Peptide bond
ing bond
Peptide bond
Peptide bond
Polyaniline
3-Triethoxysilylpropylamine, [83,85], and amino-silane
None
Glutaraldehyde modified surfaces [20,81,82].
Antibody Similar
opening strategy was reported for
Polytyramine
Mercaptopropylmethyldimethoxysilane
Polytyramine the The Glutaraldehyde
immobilization
Glutaraldehyde
Glutaraldehyde
N-y-maleimidobutyryloxy
mide ester
different methods
of phages
succini-
succini-
for
to a
Antibody
Antibody
Antibody
Antibody
Antibody
covalently
polytyramine
Peptide
AntibodyPeptide
Peptide
Antibody Peptide
Epoxide
conjugating
insulating
bond
Peptide
Peptide
bondbondbond
Peptide
bond for
ring-open-
biomolecules
layer
bond
the [81] [65]
bondto insulating
capacitive [60] layers
detec-
Polyaniline
Mercaptopropylmethyldimethoxysilane linoethyl)carbodiimide
N-Cyclohexyl-N′-(2-morpho- Antibody Peptide bond [81]
Mercaptopropylmethyldimethoxysilane mide
mide ester
None
ester Antibody ing [81]
Aminobutyldimethylmethoxysilane are now
tion summarized
of Salmonella spp. inNoticeably,
EDC/NHS Table 2.
N-Cyclohexyl-N′-(2-morpho- the sensor could be used up tobond
Peptide 40 [81]
times following al-
3-Triethoxysilylpropylamine, linoethyl)carbodiimide Aptamer Epoxide ring-
kaline treatment to
None regenerate the active ssDNA
sensing
Antibody surface Peptide
[76]. Other bond
cross-linkers such as
Polyaniline
linoethyl)carbodiimide opening [83][55] [81]
3-Triethoxysilylpropylamine Table 2. Methods
N--maleimidobutyryloxy reported for the covalent
succinimide conjugation Epoxide
of capturering-open-
biomolecules
ester [41,55] or dithiobis (succinimidyl [85]on insulating lay-
propio-
None
Glutaraldehyde Antibody Peptide ing [41]
ers. R: [47]
Capture molecule.
None Antibody
Antibody Peptide bondbond sensing [76][41]
3-Triethoxysilylpropylamine, nate) were
Glutaraldehyde None
disclosed None for the
succini-conjugation
Antibody of Epoxide
Abs to
Peptide ring-open-
capacitive
bond surfaces.
[41]
Polyaniline
Mercaptopropylmethyldimethoxysilane None
N-y-maleimidobutyloxy
Glutaraldehyde succinimide Antibody Peptide bondring- [81][55]
Epoxide
Polyaniline
Aminobutyldimethylmethoxysilane mide ester Peptide
Epoxide
Capture
ing
Epoxide
Epoxide
ring-open-
bond ring-open-
ring-opening [19]
Mercaptopropyltrimethoxysilane N-y-maleimidobutyloxy
N-y-maleimidobutyloxy ester succinimide
succinimide Antibody
Phage Antibody
AntibodyAntibody Peptide bond Linkage
opening [65]
Function Introduced GlutaraldehydeCoupling
N-y-maleimidobutyryloxy Agent
succini- Antibody
Antibody Peptide Chemical
Peptidebondbond ing Reference
Mercaptopropyltrimethoxysilane
Polytyramine
ester for covalently conjugating
Molecule ing biomolecules to insulating layers
Mercaptopropylmethyldimethoxysilane N-Cyclohexyl-N′-(2-morpho-
are now summarized
ester
mide esterin Table 2. Antibody Peptide bond[81] [55] [60]
Aminobutyldimethylmethoxysilane linoethyl)carbodiimide
None Peptide bond [55]
Glycidoxypropyldimethylethoxysilane EDC/NHS
N-Cyclohexyl-N′-(2-morpho- Aptamer Peptide bond [20]
3-Triethoxysilylpropylamine N-y-maleimidobutyryloxy
None Antibody Peptide
Epoxide bond[83][41]
ring-
3-Triethoxysilylpropylamine N-y-maleimidobutyryloxy linoethyl)carbodiimide ssDNA [55]insulating lay-
Table 2. Methods Glutaraldehyde
reported
succinimide forsuccini-
ester the covalentAntibody
Antibody
conjugation ofPeptide
Epoxide
Antibody capture bond
Peptide bond on
biomolecules
ring-open-
opening
[47]
Mercaptopropylmethyldimethoxysilane Glutaraldehyde
ers. R: Glutaraldehyde
Capture molecule.
mide ester succinimide Antibody
Antibody Peptide
Peptide bondbond [85][47]
[47][55][55]
[55]
N-y-maleimidobutyryloxy succini- Antibody Di- Antibody Peptide ing
Peptide bondbond [55] [86]
Mercaptopropyltrimethoxysilane None [41]
Polyaniline
Polydimethylsiloxane ester propionate)
thiobisGlutaraldehyde
(succinimidyl Di- Antibody
Antibody
Di- Antibody
Antibody Peptide
Peptide bondbond
Peptide bond[81]
3-Triethoxysilylpropylamine, mide ester
succini- succini-
N-y-maleimidobutyryloxy succinimide AntibodyAntibody
Antibody Peptide
Peptide
Epoxide
Capture bondbond
Peptide
Peptide
ring-open- bond bond
Polyaniline
Function
Polydimethylsiloxane Introduced thiobis
thiobis (succinimidyl
N-y-maleimidobutyloxy propionate)
succinimide
Coupling
(succinimidyl Agent
propionate) Antibody Peptide bond Linkage
Chemical Reference
Polydimethylsiloxane
Mercaptopropylmethyldimethoxysilane ester mide
succinimide
mide ester ester
ester Antibody Antibody
Molecule ing Peptide bond [65]
Glycidoxypropyldimethylethoxysilane ester
linoethyl)carbodiimide [60]
3-Triethoxysilylpropylamine N-Cyclohexyl-N′-(2-morpho- [41] [55]
Mercaptopropylmethyldimethoxysilane N-Cyclohexyl-N′-(2-morpho-
linoethyl)carbodiimide Peptide bond[55][47] [20]
N-y-maleimidobutyloxy succinimide Aptamer
Antibody
Antibody ssDNAPeptide Peptide
bondbond
Peptide bond [41]
None Di- Antibody Peptide bond
Mercaptopropyltrimethoxysilane N-y-maleimidobutyryloxy succini-
Glutaraldehyde
succinimide ester Antibody Peptide bond bond[83]
Peptide
3-TriethoxysilylpropylamineN-y-maleimidobutyloxy
ester
thiobisGlutaraldehyde
(succinimidyl
Epoxide
Antibody
Antibody Peptide
ring-open-
Peptide bond [41][47][41] [41]
[41]
Polydimethylsiloxane mide ester propionate)
ing
bond
bond [55] [86]
Mercaptopropyltrimethoxysilane Antibody Peptide bond [55]
3-Triethoxysilylpropylamine, ester
N-y-maleimidobutyloxy succinimide Antibody
Polyaniline N-y-maleimidobutyloxy
succinimide
succinimide
succini-
succinimide AntibodyPeptide
Di- Antibody
Antibody
Antibody
Antibody Peptide
bond
Peptide
Peptidebondbondbond
Mercaptopropyltrimethoxysilane thiobis ester
(succinimidyl propionate) Peptide bond
Mercaptopropylmethyldimethoxysilane ester
ester Antibody
Mercaptopropyltrimethoxysilane mide esterester
succinimide ester Antibody Peptide bond[41][47][41][60]
Mercaptopropyltrimethoxysilane EDC/NHS Di- Antibody Peptide bond [55][47] [20]
3-Triethoxysilylpropylamine N-y-maleimidobutyloxy succinimide AntibodyPeptide Peptide
bond bond
Polydimethylsiloxane N-y-maleimidobutyloxy
None
thiobis (succinimidyl succinimide
propionate) Antibody Peptide bond [47]
Glutaraldehyde Di- Antibody ssDNA Peptide
Peptide bondbond bond[47]
Mercaptopropyltrimethoxysilane ester esterpropionate)
thiobis (succinimidyl
succini- Antibody Epoxide
Antibody Peptide
ring-open- [41]
Mercaptopropylmethyldimethoxysilane Di- Di-
Antibody Antibody ingbondPeptide bond [41]
mide ester
Antibody Peptide
Peptide bond
Mercaptopropyltrimethoxysilane thiobis (succinimidyl
N-y-maleimidobutyloxy succinimide Antibody
propionate) Peptide bond
3-Triethoxysilylpropylamine, thiobis (succinimidyl propionate) Peptide bond [19]
Mercaptopropyltrimethoxysilane N-y-maleimidobutyloxy succinimide Antibody [81]
ester
Aminobutyldimethylmethoxysilane ester [65]
N-Cyclohexyl-N′-(2-morpho- Antibody Peptide bond[47] [47]
EDC/NHSDi-
linoethyl)carbodiimideDi- Antibody
Antibody Peptide Peptide
bond
Peptide bond bond[41]
[55] [41]
Aptamer [20]
thiobis (succinimidyl propionate) Peptide Peptide
bond
3-Triethoxysilylpropylamine thiobis (succinimidyl
None
propionate)
succinimide
succini- Antibody Antibody Peptide bond bond [47]
Mercaptopropyltrimethoxysilane ester Glutaraldehyde Peptide bond
Epoxide ring-open-
Antibody
Mercaptopropylmethyldimethoxysilane mide ester ester Di- Antibody
Antibody Peptide bond [47]
Polydimethylsiloxane thiobis (succinimidyl propionate) Di- ing
Polydimethylsiloxane Antibody Peptide bond
N--maleimidobutyryloxy succinimide ester [41,55] or dithiobis (succinimidyl propio-
nate) [47] were disclosed for the conjugation of Abs to capacitive sensing surfaces.
[55]
The different methodssuccini- Antibody
for covalently Peptide
conjugating bond
biomolecules to insulating layers
Mercaptopropylmethyldimethoxysilaneare now summarized [41]
mide esterin Table 2.
Biosensors 2023, 13, 17 15 of 22
N-y-maleimidobutyloxy succinimide Antibody Peptide bond
Table 2. Methods reported
ester for the covalent conjugation of capture biomolecules on insulating lay-
ers. R: Capture molecule.
[41]
MercaptopropyltrimethoxysilaneTable 2. Cont.
AntibodyCapturePeptide bond
Function Introduced N-y-maleimidobutyloxy
Couplingsuccinimide
Agent Chemical Linkage Reference
Function Introduced
Coupling Agent Molecule
Capture Molecule Chemical Linkage Reference
ester
[47][47] [47]
[86]
Di-Di- Antibody
Dithiobis Antibody Peptide
bond
Peptide bond
Polydimethylsiloxane thiobis N-Cyclohexyl-N′-(2-morpho-
(succinimidyl
(succinimidyl
thiobis propionate)
(succinimidyl propionate)
propionate) Antibody Peptide bond
However, no mention of click chemistry, or use of vinyl sulfone moieties were found
inAs
thealternative
literature for the conjugation
chemical linkage toof peptide-bond
biomolecules to capacitivea sensing
formation, surfaces,
few other types while
of
encountered in the context of EIS detection [91–93].
chemical conjugations were reported in the context of the fabrication of capacitive
[19]
biosensors. When considering nucleic acids as capture molecules, phospharamidite bond
4.2.3. Influence of the Conjugation Strategy on the Sensor Performance
formation is of interest for their covalent immobilization.
Antibody Liao andbond
Peptide Cui reported the
attachmentThe immobilization
of a strategy has
phosphatedbeen highlighted
aptameras a crucial
with parameter to consider
1-Ethyl-3-(3-
EDC/NHS
for the preparation of a capacitive sensing surface [9,13,19,52].
Castiello et al. recently reported a comparative study of four immobilization tech-
niques for the conjugation of Abs to interdigitated gold electrodes, based on peptide
coupling to: (i) a mercapto-alkyl SAM; (ii) an amino-silane monolayer between the elec-
trodes; or a iii) spin-coated poly(methyl methacrylate) (PMMA) layer. These [65] covalent
strategies were assessed against passive adsorption of the probe on the electrode (Figure 6).
Immobilizationlinoethyl)carbodiimide
to a PMMA spin coated electrode providedPeptide bond
the best capacitive behavior
Aptamer
due
3-Triethoxysilylpropylamine to its smooth surface, leading to reproducible detection of the antigen-Ab binding
events. Adsorption of Abs on the gold electrode resulted in the most heterogeneous surface
covering and the least repeatable measurements. Additionally, the immobilization layer
configuration—on electrodes only (SAM layer, B), in between the electrodes only (APTES
modification, C) or on both (PMMA layer, A)—impacted the overall performance [60]of the
sensing device. The binding events occurring in between the electrodes were playing a
major role in the overall change in capacitance
compared Peptide
ssDNA to the ones
bondoccurring directly
at
the surface of the electrodes [19]. Therefore, we believe that the SAM deposition of the
capture molecule (via the use of a linker or not) on interdigitated electrodes should not be
indicated as it leads to the deposition of the probe only at the top of the electrodes.
4.3. Impact of Surface Cleanliness and Contamination

Significant variations in reported results among different studies may arise [20]from the
lack of homogeneity and reproducibility
Glutaraldehyde of the coating
Antibody procedure, as a
Peptide bond result of improper
electrode cleaning before functionalization [4,94]. In the context of mercapto-alkyl SAM
formation on gold electrodes, Love et al. discussed the importance of proper electrode
cleaning procedures to achieve uniform coatings [18]. SAM formation is based on exchange
process, suggesting that thiolated molecules can displace miscellaneous contaminants ad-
sorbed at the electrode surface. However, the presence of contaminants greatly affects the
kinetics of the reaction, and therefore its reproducibility. To achieve reproducible coatings,
the electrodes can be cleaned with piranha solutions or oxygen plasma [18], or via electro-
chemical methods [14] in the case of metallic electrodes. In 2010, Bhalla et al. compared the
efficacy of piranha, plasma, reductive and oxidative cleaning methods on micro-fabricated
chips used for EIS detection [94]. By analyzing cyclic voltammetry scans, scanning electron
microscope images and capacitance measurements, the authors demonstrated that the
two electrochemical cleaning techniques could effectively remove contaminants from the
chips without degradation. However, the reductive pathway may lead to the deposition of
materials on the conducting surface. Therefore, oxidative electrochemical treatment was
found to be the most suitable and reproducible method for cleaning gold electrodes [94].
Biosensors 2023,13,
Biosensors2023, 13,17
x FOR PEER REVIEW 16 of
15 of 22
22
Figure 6. Schematic representation of the different immobilization strategies and architectures for
Figure 6. Schematic representation of the different immobilization strategies and architectures for
the
the conjugation
conjugation of
of insulin
insulin on
on IDEs.
IDEs. (A)
(A) spin-coated
spin-coated PMMA
PMMA deposition;
deposition; (B)
(B) mercapto-alkyl
mercapto-alkyl SAMSAM
deposition,
deposition, (C) APTES covering; and (D) passive adsorption. Adapted from Castielloet
(C) APTES covering; and (D) passive adsorption. Adapted from Castiello etal.
al.[19].
[19].
4.4. Non-Specific Adsorption
4.3. Impact of Surface Cleanliness and Contamination
Non-specific adsorption of molecules has a critical impact on biosensing measure-
ments, Significant
especiallyvariations
when using incapacitive
reported results
detection among different
[10,62]. studies may
Any molecule arise from
immobilized the
at the
lack of homogeneity and reproducibility of the coating procedure,
surface of a capacitive sensor through non-specific interactions may result in false-positive as a result of improper
electrode cleaning
detection, beforegreatly
and therefore functionalization [4,94]. In the
reduces its selectivity. Suchcontext
matrixof mercapto-alkyl
effect was described SAM
formation on gold electrodes, Love et al. discussed the importance
by Liao and Cui, in the context of capacitive detection of platelet-derived growth factor. of proper electrode
cleaning
The studyprocedures
demonstrated to achieve uniform
the beneficial coatings
effect [18]. SAM
of electrode formation
potential sweeping is based on ex-
in potentio-
change process, suggesting that thiolated molecules can displace
static EIS for discriminating between specific target binding and non-specific adsorption of miscellaneous contami-
nants adsorbed
biomolecules at the
at the electrode
surface of the surface.
sensorsHowever, the presence
[65]. Although, despiteofthis
contaminants
optimization, greatly
the
affects
ratio of the
the kinetics
positive of to the reaction,
negative andwas
control therefore its reproducibility.
still around To achieve
10:1. By increasing reproduc-
the background
ible coatings,
noise, the matrix theeffect
electrodes
can alsocandecrease
be cleaned the with piranha
sensitivity of solutions
the studied ordevice.
oxygenFor plasma [18],
example,
or via electrochemical methods [14] in the case of metallic electrodes.
the detection of Herpes virus 1 reached a LoD value of 0.21 fM in neat serum while the In 2010, Bhalla et al.
compared the efficacy of piranha, plasma,
attomolar detection range was achieved in pure buffer [33]. reductive and oxidative cleaning methods on
micro-fabricated chips used for strategies—classified
A variety of anti-biofouling EIS detection [94]. Byasanalyzingactive or cyclic
passivevoltammetry
techniques—were scans,
scanning electron
explored for many microscope
biomedical images and capacitance
applications, such asmeasurements, the authors
bioelectronic devices, demon-
biosensors,
strated that thedental
nanoparticles, two electrochemical cleaning techniques
implants or polymeric materials could
[95–99].effectively
Physical remove contam-
and chemical
inants from
passive the chips
methods includewithout degradation.
the addition However,blocking
of adsorption the reductive
agentspathway
and the mayadditionlead of to
athe deposition
repelling of materials
chemical layer basedon theon conducting
a polyethylene surface. Therefore,
glycol oxidative
(PEG) layer, electrochemi-
alkanethiol SAM
cal treatment
layer, was found
or zwitterionic polymer. to beControlling
the most suitable
the extentandofreproducible method formay
biomolecule adsorption cleaning
also
gold
be electrodes
achieved [94].
by changing the surface topography. Active methods, on the other side, create
shear forces that are stronger than the forces causing non-specific adsorption. They can be
4.4. Non-Specific
generated through Adsorption
acoustic waves generation, pressure-driven flow, or from electrical or
mechanical transducers
Non-specific adsorption [95]. Toof our knowledge,
molecules has a only passive
critical impactmethods were reported
on biosensing measure- to
reduce non-specific adsorption of biomolecules on capacitive
ments, especially when using capacitive detection [10,62]. Any molecule immobilized at sensing surfaces.
Among of
the surface physical methods,
a capacitive the addition
sensor through of bovine serum
non-specific albumin may
interactions (BSA)result
as a blocking
in false-
agent wasdetection,
positive reported for and thetherefore
detectiongreatly
of enzymes
reduces[35],its
Abs [34], disease
selectivity. protein
Such matrixmarkers
effect[8,14,67],
was de-
viruses [26,32] or cells [73]. The use of biotin [74] or glycine [53]
scribed by Liao and Cui, in the context of capacitive detection of platelet-derived growth was shown to minimize
non-specific binding
factor. The study events. Additionally,
demonstrated the addition
the beneficial effect ofof concentrated
electrode solutions
potential of KClinwas
sweeping po-
found to greatly reduce non-specific adsorption by disrupting
tentiostatic EIS for discriminating between specific target binding and non-specific weak interactions. Dijskma
Biosensors 2023, 13, 17 17 of 22
et al. showed that the injection of 100 mM KCl solution completely remove interferon
gamma from gold surface without damaging the SAM functionalized layer [75].
Among chemical methods, the addition of an anti-biofouling PEG layer was high-
lighted in DNA-hybridization and interleukin biosensors [57,76]. Miranda-Figueroa et al.
demonstrated the beneficial effect of added PEG chains on malaria biosensors. Not only
the matrix tolerance was improved, but also the LoD adynamic detection range were
enhanced [30].
The design of suitable strategies against nonspecific binding highly depends on the
nature of the targeted analytes, therefore requiring extensive trial iterations. Dykstra et al.
developed a microfluidic platform that can measure protein adsorption on selected surfaces.
This device offers the possibility to rapidly screen various materials toward their tendency
to repel biomolecules, and could be of great interest for the design of capacitive biosensors
in the future [100].
4.5. Surface Insulation and Coverage

The surface of capacitive sensors must be insulated and hole-free to avoid charges
to move through the layer, leading to the apparition of a faradaic current between the
conductors [3,19,20,71], that would result in a change of capacitance of the surface and
therefore a decrease in sensitivity [10]. Common insulating strategies relies on SAM
covering based on alkyl-thiols, polymeric layers and silanization [19].
In the context of gold-thiol SAM formation, alkylthiols are added to insulate the
sensing layer. Mirsky et al. reported that long chains should be privileged as short
chains were prone to desorption. Proper insulation of gold electrodes was achieved with
15-/16-mercaptohexadecanoic acid [73]. Later, dodecanethiol [21,39,40,76–78,88], hexade-
canethiol [101], mercaptohexanol [102], and mercapto-undecanol [4] were extensively used
to insulate gold electrodes.
The quality of surface insulation largely depends on the selection of the functionaliza-
tion procedure. Rickert et al. studied the insulation of epitope-modified gold electrodes
with hydroxyundecanethiol (HUT). Simultaneous adsorption of a mixture of HUT and
peptide was compared to the sequential adsorption of both components. The adsorption
of mixed solutions resulted in poorly reproducible functionalization. On the contrary,
reproducible and highly resistive films were obtained when the HUT was adsorbed after
the epitope was immobilized [28].
In addition to provide chemical functionalities for the post-conjugation of capture
biomolecules, polymeric layers were reported for the insulation of conductive electrodes.
The insulation of Abs-modified gold electrodes with a 50 nm polytyramine film led to the
detection of HSA down to 1.6 ng/mL concentration and with high reproducibility [72]. The
quality of the insulating layer was probed by cyclic voltammetry. Berney et al. developed
a capacitive detector for transferrin and studied the effect of PEG, as a non-conductive
polymer, to insulate the sensing surface. When transferrin Ab was immobilized on non-
insulated surface, the capacitance measurements after exposition to the targeted antigen
were not reproducible. The addition of a PEG overlay system indicated the possibility to
develop differential capacitive biosensors. However, the lack of continuity and integrity of
the PEG layer did not allow for quantitative measurement of transferrin [20].
In conclusion, several requirements must be followed when designing and preparing
a sensing surface for capacitive biosensors. First, the surface must be free of contaminants
and prepared in as clean conditions as possible. Then, an insulation layer must be present
to avoid faradaic currents that would lead to a drastic decrease in sensitivity. In the case
of deposition of an oxide or polymeric layer on top of the electrodes, this layer should
be however as thin as possible to keep good sensitivity properties. Finally, non-specific
adsorption should be avoided to reduce false-positive results. Toward this goal, the addition
of BSA or PEG layers have been the most reported technique.
Biosensors 2023, 13, 17 18 of 22
5. Conclusions and Perspectives

Capacitive biosensors could greatly contribute to various diagnostic applications.
Compared to other classic sensors, they offer the opportunity to develop in rapid and
non-expensive portable sensors. Since their first development 30 years ago, capacitive
biosensors were mainly applied to virus detection, and cancer or inflammatory diseases
diagnostics. Several limitations restrained their expansion to other clinical applications. In
particular, non-faradaic impedimetric sensors generally suffered from poor measurements
reproducibility [4,12,13,19] and large standard deviations, as a result of non-optimal surface
characteristics. Among the parameters which were evaluated to improve the performance
of capacitive biosensors, one might concentrate on the following aspects: (i) surface cleaning
by electrochemical treatment proved to increase the repeatability of subsequent function-
alization steps; (ii) selection of the immobilization strategy in combination with proper
insulation techniques showed to have drastic impact on the resulting sensor sensitivity;
and (iii) the addition of blocking agents to mitigate non-specific adsorption events resulted
in enhanced specificity and sensitivity.
Considering these multiple parameters, we can expect that the next generation of ca-
pacitive biosensors will result from rational design of the surface composition, morphology
and geometry to enlarge the scope of these sensors in clinical applications.
Author Contributions: Conceptualization, analysis, original draft preparation, review and editing,
P.R. Review and editing, project administration, funding acquisition, S.G.-L. All authors have read
and agreed to the published version of the manuscript.
Funding: This review was prepared in the frame of the projects supported by the Swiss National
Science Foundation (NRP78, Grant n◦ 4078P0_198265) and Innosuisse (Innovation project DeMoViS,
Grant n◦ 38934.1 IP-LS).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: We do not present original data in the manuscript as it is a review article.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the
writing of the manuscript or in the decision to publish this review.
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biosensors

Keywords: non-faradaic impedance; capacitive detection; biosensors; electrochemical impedance

Biosensors 2023, 13, 17. https://doi.org/10.3390/bios13010017 https://www.mdpi.com/journal/biosensors

Figure 1. Illustration of the different types of electrical sensors.

2.1. Potentiostatic Capacitance

Figure 2. Schematic representation of electrode–solution interfaces for capacitive detection of

Biosensors 2023, 13, 17 5 of 22

3.3. Unicellular Organisms

Unicellular organisms Carboxylic acids introduced via

4. Capacitive Sensing Surfaces Preparation, and Limitations

4.2. Biomolecules Immobilization Techniques

4.2.1. Self-Assembled Monolayer (SAM) Formation on Metal Electrodes

4.3. Impact of Surface Cleanliness and Contamination

4.5. Surface Insulation and Coverage

5. Conclusions and Perspectives

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