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Deep Eutectic Solvents (DESs) and Their Application in
Rossella Svigelj *, NicolòDossi, Cristian Grazioli and Rosanna Toniolo *
Citation: Svigelj, R.; Dossi, N.;
Grazioli, C.; Toniolo, R. Deep Eutectic
Solvents (DESs) and Their
Application in Biosensor
Development. Sensors 2021,21, 4263.
Academic Editor: Arnaud Buhot
Received: 6 May 2021
Accepted: 17 June 2021
Published: 22 June 2021
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Department of Agrifood, Environmental and Animal Science, University of Udine, Via Cotoniﬁcio 108,
33100 Udine, Italy; email@example.com (N.D.); firstname.lastname@example.org (C.G.)
*Correspondence: email@example.com (R.S.); firstname.lastname@example.org (R.T.)
Deep Eutectic Solvents (DESs) are a new class of solvents characterized by a remarkable
decrease in melting point compared to those of the starting components. The eutectic mixtures can
be simply prepared by mixing a Hydrogen Bond Acceptor (HBA) with a Hydrogen Bond Donor
(HBD) at a temperature of about 80
C. They have found applications in different research ﬁelds; for
instance, they have been employed in organic synthesis, electrochemistry, and bio-catalysis, showing
improved biodegradability and lower toxicity compared to other solvents. Herein, we review the use
of DESs in biosensor development. We consider the emerging interest in different ﬁelds of this green
class of solvents and the possibility of their use for the improvement of biosensor performance. We
point out some promising examples of approaches for the assembly of biosensors exploiting their
compelling characteristics. Furthermore, the extensive ability of DESs to solubilize a wide range of
molecules provides the possibility to set up new devices, even for analytes that are usually insoluble
and difﬁcult to quantify.
Keywords: deep eutectic solvents; biosensors; DNA; enzymes; MIPs; aptamers; nanomaterials
1.1. Deep Eutectic Solvents (DESs)
Lately, a new class of solvents, called Deep Eutectic Solvents (DESs), has been success-
fully employed in different research ﬁelds. In 2004, Abbott et al. described the generation
of eutectic mixtures as complexes formed between halide salts and a range of compounds
such as amides and carboxylic acids [
]. Indeed, DESs can be easily prepared by mixing at
least two components able to form a eutectic mixture with a melting point lower than that
of the starting components [
]. This group of solvents share some characteristics with
ionic liquids (ILs), a class of organic salts with a low melting point, generally obtained by
combining an organic cation (commonly imidazolium-based cations) with a large variety
of anions (i.e., Cl
]. However, DESs have demonstrated to have
some interesting advantages over ILs. As a matter of fact, the deﬁnition of ILs as “green”
species is largely contested in the current literature [
]. Therefore, to overcome the high
cost of their production and their toxicity, DESs have arisen as a cheaper and greener
Recently, another class of solvents, called Natural Deep Eutectic Solvents (NADESs),
has been introduced [
]. This new category was described after observing the occurrence of
certain natural mixtures. In fact, the presence of eutectic liquids in living organisms could
explain a great number of biological processes where poorly water-soluble metabolites
are involved [
]. In different types of microbial, animal, and plant cells, the presence of
substances, including sugars, some amino acids, choline, and some organic acids such as
malic acid, citric acid, and lactic acid, could lead to the formation of NADESs as a third type
of solvent besides water and lipids. Furthermore, the existence of these natural solvents
could explain the survival of some organisms through extreme conditions such as cold or
water shortage .
Sensors 2021,21, 4263. https://doi.org/10.3390/s21134263 https://www.mdpi.com/journal/sensors
Sensors 2021,21, 4263 2 of 18
DESs are usually prepared by mixing a Hydrogen Bond Acceptor (HBA) with a
Hydrogen Bond Donor (HBD) at a temperature of about 80
C. Among the HBAs, different
quaternary ammonium salts can be found, while amines, carboxylic acids, alcohols, or
carbohydrates belong to the group of HBDs (Figure 1). DESs have found applications in
various research areas, including organic synthesis, electrochemistry, and bio-catalysis [
Furthermore, they display improved biodegradability and lower toxicity compared to other
solvents, characteristics that make them particularly interesting in green chemistry .
Figure 1. Typical HBDs and HBAs employed in DESs.
Interestingly, both DESs and NADESs provide a network of hydrogen bonds that
make possible the solubilization of a wide range of molecules [
]. Their characteristics
make them potential universal solvents capable of extracting a wide variety of non-polar
and polar compounds [
]. Thus, they could become an appealing alternative to several
standard and toxic organic solvents. The structures of their components strongly inﬂuence
all DESs’ physicochemical properties, such as melting point, density, conductivity, and
]. Moreover, studies on the three-dimensional structure of DESs revealed that
the interaction between HBDs and HBAs depends mainly on hydrogen bonds, but they
also interact with the anion by arranging themselves around it [
]. In general, the presence
of a broad hydrogen-bonding network among the components is responsible for the high
viscosity of DESs and the restrained mobility of free species inside the solvent. Additional
phenomena, such as van der Waals and electrostatic interactions, may also be responsible
for the high viscosity of DESs. The freezing point of DESs is characterized by a consistent
drop, generally wider than 150
C. The difference in the freezing point of a binary mixture
Sensors 2021,21, 4263 3 of 18
in the eutectic composition compared to that of a theoretical ideal mixture is related to
the magnitude of the interaction of the components [
]. DESs exhibit, in general, higher
densities than water, with values ranging from 1.041 g cm
to 1.63 g cm
]. Among the
different quaternary ammonium salts used in eutectic mixtures, choline chloride (ChCl) is
the most employed thanks to its low cost and biodegradability. ChCl can be combined with
HBDs to provide DESs with different physical and chemical properties such as freezing
point, viscosity, conductivity, and pH [
]. Table 1shows some physical parameters of
Table 1. Physical parameters of some ChCl-based DESs.
DESs Salt/HBD Molar Ratio Viscosity aPa·s Conductivity aµS·cm−1Density ag/cm3Tf/◦C
ChCl:urea 1:2 - - 1.25 12
ChCl:acetamide 1:2 0.127 2710 1.09 51
ChCl:glycerol 1:2 0.177 1647 1.19 −40
ChCl:1,4-butanediol 1:4 0.047 2430 1.04 -
ChCl:triethylene glycol 1:4 0.044 1858 1.12 −66
ChCl:xylitol 1:1 3.867 172.6 1.24 -
ChCl:D-sorbitol 1:1 13.736 63.3 1.28 -
ChCl:oxalic acid 1:1 0.089 2350 1.24 34
ChCl:levulinic acid 1:2 0.119 1422 1.13 -
ChCl:malonic acid 1:1 0.616 732 1.21 10
ChCl:malic acid 1:1 11.475 41.4 1.28 -
ChCl:citric acid 1:1 45.008 18.4 1.33 69
ChCl:tartaric acid 2:1 66.441 14.3 1.27 47
aValues determined at 30 ◦C; Tf= freezing point.
To date, DESs have found many applications in analytical chemistry, such as in the
extraction of analytes from complex liquid and solid matrices [
], as modiﬁcation media
for nanomaterials [
], for elution in dispersive solid-phase extractions, and as a mobile-
phase modiﬁer in chromatography [
]. They have also been employed in the extraction
of bioactive compounds [
], including ﬂavonoids, phenolic acids, polyphenols, saponins,
and anthraquinones, from various types of natural sources [
]. In addition, DESs
display a good capability of solubilizing many other compounds, such as drugs, metal
oxides, and carbon dioxide [24,25].
Taking into account all their properties and their biocompatibility, this class of solvents
could replace the classic aqueous buffers that are normally used in the development of
biosensors. DESs could also improve biosensor performance, for example, in the analysis
of molecules that are not soluble in water.
Here we discuss the recent uses of DESs in biosensor development and their combi-
nation with different materials to enhance the sensing performance of biobased sensing
devices, underlining the advantages that these solvents bring to the construction of minia-
turized and green biosensors (Figure 2). DESs have been used for the generation of
nanomaterials and the modiﬁcation of electrodes with graphene, carbon paste, and carbon
nanotubes. Furthermore, DESs stabilize DNA molecules, favoring the development of
stable biosensors; they increase the activity of some enzymes, providing better sensitivity;
and ﬁnally, they can favor the creation of MIPs for poorly soluble molecules.
Biosensors exploit the sensitivity of transducers combined with the high speciﬁcity of
biological recognition elements, which are able to interact selectively with analytes [
Generally, biosensors can be categorized on the basis of their transduction mechanism,
which can be optical (including optical ﬁber and surface plasmon resonance biosensors),
Sensors 2021,21, 4263 4 of 18
electrochemical (including voltammetric, amperometric, and impedance biosensors), or
piezoelectric (including quartz crystal microbalance biosensors) [
]. Alternatively, their
classiﬁcation is based on the sensing element, in which case they are named immunosensors,
aptasensors, genosensors, enzymatic biosensors, and molecularly imprinted sensors when
the biological sensing elements are antibodies, aptamers, nucleic acids, enzymes, and MIPs
(molecularly imprinted polymers), respectively (Figure 3) [
]. The impact of biosensing
is gaining importance in all sectors, such as clinical, environmental, and food-related
]. Biosensors can be considered very versatile and powerful tools because of
their low cost and compatibility with portable and compact instrumentation, as well as
their easy and rapid use.
Electrochemical biosensing platforms arise from the combination of different disci-
plines such as chemistry, material science, biology, and electronic engineering. A signiﬁcant
example of successful electrochemical biosensing is the glucose sensor that relies on the
interaction of the analyte with an enzyme (glucose oxidase), giving rise to a current directly
proportional to the blood glucose level [
]. In the last years, great progress has been
made: for instance, a continuous glucose monitoring system has been introduced that
allows better control of a patient’s blood glucose levels [
]. This technology is based
on implantable transmitters detecting blood sugar levels directly in the body. With the
same purpose, wearable electrochemical sensors have been developed which integrate, in
a non-invasive and non-obtrusive way, sensing systems suitable for various monitoring
applications directly onto either textile materials or the patient epidermis [33,34].
Overview of the general advantages of the use of DESs in biosensor development. The use
of DESs can improve the performance of biosensors thanks to the biocompatibility of this class of
solvents with the recognition elements and the improvement of electrode material performance.
Sensors 2021,21, 4263 5 of 18
Figure 3. Schematic representation of biosensor classiﬁcation.
All these innovations show that it is possible to develop technologies that can have
real repercussions on the market and that the demand for advanced portable and green
devices will extensively grow in the near future.
2. Synthesis of Electrode Materials in DESs
The use of DESs can improve the performance of electrode materials. Recently, some
works have reported the application of DESs in the development and use of carbon paste
electrodes and of solid electrodes modiﬁed with graphene, nanoparticles, and carbon nan-
otubes. Within the development of different materials, DESs can offer several advantages,
including avoiding the aggregation of nanoparticles and allowing an effective functional-
ization of nanotubes. Moreover, the high solubility of various types of chemicals, like metal
salts, metal oxides, solid halogens, and small organic molecules, and green characteristics
are attractive features associated with the use of DESs and explain their increasing use.
2.1. Graphene, Carbon Paste, and Carbon Nanotubes in DESs
Several materials have been modiﬁed with DESs, including graphene, carbon paste,
and nanotubes. The modiﬁcation of these materials with the eutectic mixtures has shown
several advantages, including increased charge transfer and conductivity, increased surface
area, and the generation of new functional groups.
Graphene and DESs have separately demonstrated encouraging properties in the
development of electroanalytical platforms [
]. Recently, Fuchs et al. combined the use
of graphene and DESs, studying their coadjuvant effects in electrochemistry [
considered the electrochemical performance of a centimeter-scale graphene monolayer
generated by chemical vapor deposition and afterward moved onto insulating SiO
supports in the DES ethaline formed by 1:2 ChCl and ethylene glycol. They provided a ﬁrst
approach for the use of graphene combined with a DES for electroanalytical applications,
covering also the characterization of the graphene/DES electrochemical behavior. The
authors also investigated the modiﬁcation of graphene with metal (Zn) and metalloid (Ge)
in the DES, evaluating their electrochemical performances. What emerged was that the
Sensors 2021,21, 4263 6 of 18
behavior of graphene in ethaline is close to the characteristics of glassy carbon or highly
ordered pyrolytic graphite; however, other features of the graphene/ethaline systems are
uncommon, thanks to the two-dimensional nature of graphene. Hence, this work gave the
ﬁrst framework for graphene–DES systems.
A recent work by Cariati and Buoro reported the employment of natural deep eutectic
solvents (NADESs) for the modiﬁcation of carbon paste electrodes [
]. Enhanced conduc-
tivity and charge transfer rate were noticed when the cyclic voltammograms of potassium
ferrocyanide were recorded with the NADES-modiﬁed carbon paste electrode compared to
the bare carbon paste, revealing smaller peak potential separation. An improvement in the
diffusion of the probe to the graphite surface was achieved with the integration of KCl in
the NADES solution, decreasing in this way the binder layer viscosity. Better performance
when using a reline (ChCl and urea, 1:2) modiﬁed carbon paste electrode was also noted
for the oxidation of dopamine and ascorbic acid.
Further, Ibrahim et al. prepared two DESs using ethylene glycol combined with N,N-
diethylethanolammonium chloride, or ChCl. In this study, the capability of DESs for carbon
nanotube functionalization was investigated by studying the changes occurring after the
functionalization process, using KMnO
as the oxidizing agent. After the modiﬁcations,
the functionalized carbon nanotubes were tested for the absorption of methyl orange from
water. The effect of DESs was an increased surface area of carbon nanotubes and the
generation of new functional groups on the carbon nanotubes’ surfaces without causing
any damage to their structure, demonstrating its usefulness in the development of modiﬁed
carbo-nanotubes for water treatment [
]. Furthermore, Rozas et al. studied the properties
of carbon, boron nitride, silicon, germanium, and molybdenum disulﬁde nanotubes in
reline by applying classical molecular dynamics simulations [
]. In this study, the inter-
actions between reline and nanotubes revealed the development of a strongly adsorbed
layer, demonstrating a great afﬁnity of reline for the considered nanotubes and, among
them, especially for the boron nitride one. The authors described a sustainable DES-based
nanoﬂuid system applicable to many different research ﬁelds. The nanotube size effect was
also studied and, in every case, reline was able to solvate the nanotubes, demonstrating
that nanoﬂuids based on reline can be synthesized for a wide range of nanotube sizes and
chemical compositions for different nanotechnology applications.
2.2. Nano and Magnetic Particles in DESs
Nanoparticles and magnetic particles have proved their importance in the develop-
ment of different analytical platforms. Although they present several advantages, among
the disadvantages are their aggregation and lack of stability over time; regarding these
aspects, the use of eutectic liquids could help expand their use.
In 2008, Liao et al. reported the synthesis of gold nanoparticles (AuNPs) with diverse
and unique shapes such as stars, snowﬂakes, and thorns using reline [
]. The authors
obtained star-shaped AuNPs using HAuCl
and l-ascorbic acid in DES at room temperature.
They outlined that the water content present in the DES can modulate different shapes.
SEM images illustrated pentagonal star-shaped AuNPs of about 300 nm; the images also
revealed the presence of three- and four-branch star-shaped nanoparticles. The fact that
DESs can inﬂuence the shape of nanoparticles could lead to progress in the synthetic
approaches for nanomaterials.
In 2014, another synthesis and growth mechanism of AuNPs in DES was described [
In this study, a low-energy, soft-sputtering deposition technique was employed for gold
nanoparticle formation in DES. The authors reported that DES can act as a structure-
directing agent in the self-assembly (SA) of gold nanoparticles, and this feature can be
modulated by anions, cations, and neutral components, such as urea. In addition, high
concentrations of AuNPs in the eutectic mixture resulted in SA in the ﬁrst and second shell
short-range ordering of the gold nanoparticles. The growth and the ordering of AuNPs
occurred on the surface as well as inside the bulk DES. The SA process depended on the
nature of the components forming the eutectic mixture, and the authors employed reline,
Sensors 2021,21, 4263 7 of 18
which is based on ChCl and urea. They obtained AuNPs of 5 nm size and SA in short-range
order when they employed reline, while when using a non-templating agent such as castor
oil they obtained AuNPs but without the SA. Moreover, the use of DES ensured that the
particles remained stable, showing no aging effect, demonstrating the stabilizing effect
Svigelj et al. monitored the behavior of magnetic particles (MPs) modiﬁed with
the peptide 33-mer in buffer and in ethaline with backscattering experiments. The MPs
dispersed in ethaline remained in suspension longer than those in buffer, where they
precipitated (speed of 0.06 mm/h) after undergoing agglomeration, which was detected
as an increase in the backscattering of light in the middle zone of the measurement cell.
In the DES, the particles displayed a homogeneous distribution throughout the entire cell
and remained stable for 24 h, with no precipitation [
]. Among the advantages of the
use of DES combined with MPs, the authors highlighted the stabilization and the ease
More recently, Baby et al. employed DESs in the solid-state synthesis of phase-pure
magnesium ferrite nanoparticles that were subsequently exploited for the concomitant
detection of nitrofurantoin (NFT) and 4-nitrophenol (4-NP) [
]. Five different DESs, based
on the combination of ChCl with malonic acid, oxalic acid, urea, ethylene glycol, and
fructose, were employed in the synthesis of the magnesium ferrite nanoparticles (Figure 4).
Synthetic path of various DES-assisted MgFe
and their use in the electrochemical detection of 4-NP and NFT.
Reprinted (adapted) with permission from Baby, J.N.; Sriram, B.; Wang, S.-F.; George, M. Effect of Various Deep Eutectic Solvents
on the Sustainable Synthesis of MgFe
Nanoparticles for Simultaneous Electrochemical Determination of Nitrofurantoin
and 4-Nitrophenol. ACS Sustain. Chem. Eng. 2020,8, 1479–1486. Copyright 2020, American Chemical Society .
The authors noted that the type of DES employed in the synthesis was decisive for the
shape of the NPs. The use of DESs based on ChCl and malonic acid or oxalic acid provided
NPs with a variety of cubical and spherical shapes, whilst NPs synthesized in the DES
based on ChCl and urea consisted of cubical nanoparticles. When ChCl in combination
with ethylene glycol was used, nanospheres and spherical agglomerations were obtained,
while the use of a DES based on ChCl and fructose led to the formation of nanograins. The
electrode modiﬁed with NPs synthesized in the ChCl and fructose DES showed the highest
sensitivity and lowest detection limits and a linear range between 0 and 342.6
also an excellent selectivity and a good reproducibility.
Sensors 2021,21, 4263 8 of 18
2.3. Synthesis of Molecularly Imprinted Polymers in DESs
Usually, molecularly imprinted polymers (MIPs) are developed against a speciﬁc
target that is used as template; however, some target compounds are not suitable as
templates because of their poor solubility in common solvents. The tailoring properties
of DESs could overcome this issue, allowing the production of MIPs for a wider range
In this regard, Fu et al. obtained an MIP for caffeic acid using a ternary ChCl–caffeic
acid–ethylene glycol (ChCl–CA–EG) DES, which was employed as a template to prepare
MIPs. The speciﬁc recognition capacity of the MIPs for polyphenols was better than
those of C18, C8, or non-molecularly imprinted polymer adsorbents, and was favorably
employed for the determination of polyphenols extracted from a sample of Radix asteris [
The authors proved that transformation of an insoluble target compound in a polymeric
DES for MIP preparation and recognition is an innovative and viable strategy for further
The use of DESs in the synthesis of MIPs is a promising approach for speciﬁc recogni-
tion of proteins. As a matter of fact, the imprinting for biomacromolecules, like proteins,
represents a difﬁcult goal to achieve because of their molecular size, low mass transfer, and
complex and changeable conformation. A step towards the solution of these problems has
been taken with the introduction of DESs in the preparation of MIPs. Xu et al. designed and
synthesized a DES-based magnetic surface imprinted polymer for the selective recognition
of lysozyme . The obtained DES-Fe3O4@SiO2-MIP displayed better adsorption ability
for the template lysozyme than DES-Fe
-NIP (non-imprinted polymer). Adsorp-
tion experiments using the single reference protein, a binary protein mixture, and, ﬁnally,
a real sample were performed to prove the selectivity and speciﬁcity of the imprinted
polymer. The authors demonstrated that recognition holes are generated on the surface
-MIP during the imprinting process (Figure 5). They also proved the
reusability of the imprinted polymer, which can be regenerated with good stability.
Figure 5. Schematic illustration of the preparation of DES-Fe3O4@SiO2-MIP. Adapted with permission from .
Li et al. employed four types of DESs for the modiﬁcation of magnetic MIPs with mul-
tiple templates based on silica. Subsequently, the authors applied the imprinted polymers
for the fast and concomitant magnetic solid-phase extraction of active diterpenoids from
Salvia miltiorrhiza bunge, major isoﬂavones from Glycine max (Linn.) Merr, and catechins
from green tea [
]. The eutectic mixture based on ChCl and glycerol showed the highest
chemisorption capacity and selectivity for all the target molecules from the natural plants,
proving an excellent extraction ability.
Sensors 2021,21, 4263 9 of 18
3. Biocompatibility of DESs
3.1. DNA Stability and Behavior in DESs
DNA plays an important role in the development of biosensors. Biosensors based on
nucleic acids are developed towards the recognition of different organism strains through
hybridization (genosensors) or exploit the ability of ssDNA to fold and bind speciﬁc target
molecules (aptasensors) .
DNA is usually employed in aqueous solutions; however, its helix structure can be
affected by non-physiological temperatures, pH values, and ionic strengths. In addition,
degradation processes promoted by nucleases can lead to its chemical instability [
This factor can be a drawback in biotechnological processes based on nucleic acids. Fur-
thermore, very small water volumes vaporize immediately under open-air conditions
or at high enough temperatures; this can be a further limitation in the development of
DNA-based nanotechnologies, where extremely small volumes are usually employed.
Consequently, the use of solvents without these limitations could provide new possibilities
in the development of DNA-based devices.
It has been reported that DESs based on ChCl guarantee long-term stability of
biomolecules such as DNA and proteins [
]. Recently, Zhao et al. described that DNA
is able to form a stable duplex in reline. This interaction between DESs and DNA comes
from the electrostatic attraction between organic cations and the DNA phosphate backbone,
accompanied by hydrophobic and polar interactions between eutectic liquids and the DNA
major and minor grooves [
]. In the case of ChCl-based DESs, choline ions establish an
interaction with atoms of all areas of DNA thanks to the creation of multiple hydrogen
bond networks that are able to stabilize the DNA duplex more effectively than the sodium
ions present in aqueous buffers. As to DNA stability, cations play a more important role
than anions because cationic species are required to weaken the repulsive forces between
phosphate groups of DNA strands, while anions only establish an interaction with the
bases through hydrogen bonds .
As shown in Figure 6, choline ions have a high afﬁnity for the A–T base pair minor
groove, which displays a smaller width and more electrostatically polar environment of
this groove compared to the major groove. As can be noted, choline ions ﬁt well into the
minor groove of A–T base pairs, and as reported from simulations, this interaction persists
over time. Furthermore, the multiple hydrogen bonds formed between choline ions and
DNA atoms preserve the conformation of the A–T-rich DNA duplex.
Molecular dynamics simulation of (
) choline ions solvating the minor groove of A–T
base pairs of duplex DNA; carbon atoms of choline ions are represented in yellow, oxygen atoms of
choline ions are represented in red, and hydrogen atoms are in white. (
) Atomic-scale representation
of the DNA–choline bonds. Adapted with permission from Nakano, M.; Tateishi-Karimata, H.;
Tanaka, S.; Sugimoto, N. Choline Ion Interactions with DNA Atoms Explain Unique Stabilization
of A–T Base Pairs in DNA Duplexes: A Microscopic View. J. Phys. Chem. B
doi:10.1021/jp406647b. Copyright 2014 American Chemical Society .
Sensors 2021,21, 4263 10 of 18
Mamajanov et al. demonstrated that at least four distinct nucleic acid secondary
structures can exist in DESs; they also noted that the anhydrous character and capacity
to support natively folded DNA and protein structures make DESs appealing for the
synthesis of biopolymers [
]. Pal et al. examined the interaction between reline and
guanine-rich quadruplex thrombin-binding aptamer (TBA) at 300 K for different reline
]. They studied the conformational behavior of the aptamer in reline
using molecular dynamics simulations. The results showed that the structure and confor-
mation of the sequence were more rigid than those in the lower reline concentration. The
experimental ﬁndings conﬁrmed the structural features of TBA in anhydrous and hydrated
reline media. Overall, this study indicates that reline is suitable for long-term nucleic
acid storage. Moreover, Pal et al. also reported that the hydrogen bonds between reline
and the proto-oncogene c-kit G-quadruplex DNA are responsible for its high temperature
]. The authors studied the conformational dynamics of c-kit oncogene promoter
G-quadruplex DNA in reline in a temperature range of 300–500 K, exploiting a 10
molecular dynamics simulation. They demonstrated that reline is able to protect the c-kit
G-quadruplex DNA, and it also decelerates the thermal denaturing process.
Lastly, Svigelj et al. reported that the use of a DES in the selection of aptamers allowed
a faster enrichment of highly afﬁne sequences [
]. The authors employed ethaline to select
aptamers against gliadin, which is a poorly water-soluble fraction of gluten. The effect of
ethaline on the selection is probably due to the ability of DESs to stabilize secondary DNA
structures, combined with the high viscosity which may add further restriction during the
selection of aptamers. The development of DNA-based biosensors in DESs is discussed in
detail in Section 4.
3.2. Enzyme Activity in DESs
Advances in biocatalysis have been made by performing enzymatic bioreactions in
partly or totally nonaqueous solvents. This aspect can be particularly interesting when
working with hydrophobic substrates, since the presence of organic solvents could de-
activate or denature the employed enzyme. In biotransformation, DESs can take part
as solvents, cosolvents, or a second phase in water–DES mixtures. The most interesting
aspect of employing DESs in biocatalytic reactions is that their presence can recreate an
environment very similar to the intracellular one, playing a new role different to that of
water and lipids .
For example, the use of DESs with hydrolases has been widely investigated [
Gorke et al. reported that the rate of hydrolysis of styrene oxide catalyzed by epoxide
hydrolase increased dramatically in DES: the conversion was only 4.6% in buffer but in-
creased to 92% upon addition of 25 vol% ChCl:glycerol, with unchanged enantioselectivity
(Scheme 1) .
Scheme 1. Scheme of epoxide hydrolase activity in DES.
The use of DESs can also impede enzyme denaturation and deactivation, phenomena
that are frequently observed in conventional organic solvents, as reported in some works
with horseradish peroxidase (HRP) and hydrolases. In addition, DESs have a positive
inﬂuence on HRP stability and enhance cytochrome C activity [58,59]. As a matter of fact,
Sensors 2021,21, 4263 11 of 18
Ghobadi et al. reported an increased binding afﬁnity of H
to Bovine liver catalase when
using a ChCl-based DES .
The effect of DESs on enzymes such as laccase is also an interesting aspect. Laccase is
an oxidase that is usually employed in biosensors to detect phenolic compounds [
sensitive element of this kind of sensor is the actual enzyme with its active center, which is
able to catalyze electron transfer reactions without cofactors. The performance of enzyme
biosensors can be inﬂuenced by pH, temperature, and the activity of the enzyme [
Laccase-catalyzed reactions have been studied in 16 different DES aqueous solutions. In
their work, Toledo et al. described that while ChCl-based DESs led to a decrease in laccase
activity, the substitution of the chloride anion in the cholinium salts with other anions
signiﬁcantly promoted the laccase activity [
]. Speciﬁcally, the DES choline dihydrogen
phosphate:xylitol and all choline-dihydrogen-citrate-based DESs resulted in an improve-
ment in the activity of laccase in oxidative reactions. Moreover, the laccase activity in
these DES aqueous solutions was higher than that in aqueous solution of its individual
components and in the buffer control, indicating a cooperative effect. Lastly, it was proved
that water–DES mixtures can be employed as media for storing the enzyme with better
performance than the classic buffer control at
C, extending the activity up to 20 days.
4. Application of DESs in the Development of Biosensors
As stated in Sections 2and 3, the advantages obtained in the use of DESs for the synthe-
sis of electrode materials and the manipulation of biomolecules can be fruitfully applied to
biosensor development. Da Silva et al. proposed a biosensor with an innovative electrode
structure based on the redox polymer poly(brilliant cresyl blue) (PBCB) electrodeposited in
ethaline on a multiwalled carbon nanotube (MWCNT)-modiﬁed glassy carbon electrode
]. The authors optimized the electropolymerization in DES and performed the
corresponding ﬁlm characterization. Afterwards, they proposed its application in enzyme-
based biosensors obtained by immobilizing glucose oxidase (GOx) or tyrosinase on PBCB
/MWCNT/GCE. The authors compared three conﬁgurations, namely,
/MWCNT/GCE, GOx/aqueous PBCB/MWCNT/GCE, and
GOx/GCE, on which the same quantity of GOx was immobilized. The setup with PBCB
deposited in DES showed the best sensitivity and a limit of detection of 2.9 mM for glucose
determination by ﬁxed potential amperometry. Moreover, the same approach was used to
prepare tyrosinase biosensors. The Tyr/PBCB Ethaline-HNO
/MWCNT/GCE proved to
be the best setup for the amperometric determination of catechol, as occurred for glucose
Da Silva et al. also proposed an electrochemical enzyme inhibition biosensor for
the pesticide dichlorvos based on choline oxidase (ChOx) immobilized on PBCB ﬁlms
electrodeposited in ethaline on a MWCNT-modiﬁed GCE [
]. Superior electrochemical
performance of PBCB nanostructured ﬁlms in ethaline was reported. In fact, ethaline,
employed as a polymerization solvent for these ﬁlms, led to smoother and more compact
nanostructured surfaces compared to the ones obtained in aqueous media. This innovative
platform presented excellent analytical parameters compared to other biosensors developed
for monitoring traces of pesticides in different matrices.
More recently, another work from da Silva et al. described the development of an am-
perometric biosensor for the detection of acetylcholine (ACh) based on acetylcholinesterase
(AChE). The enzyme was immobilized on poly(neutral red) (PNR) ﬁlms generated by elec-
tropolymerization on Fe
magnetic-nanoparticle-modiﬁed GCEs (Figure 7) [
was employed for the electropolymerization of neutral red by potential cycling. Since eu-
tectic solvents have low ionic strength, ethaline was added with acid dopants (HNO
, HCl, and HClO
). The composition of the ethaline–acid solutions inﬂuenced the
morphology and, consequently, the electrochemical behavior of the polymer ﬁlm. The
biosensor was tested for the determination of ACh in synthetic urine and exhibited good
selectivity, reproducibility, stability, and high selectivity, with rapid response and low limit
Sensors 2021,21, 4263 12 of 18
of detection. The good results obtained for the analyte open the possibility to use this
biosensor in clinical diagnosis.
Schematic representation of the biosensor for acetylcholine detection. The glassy carbon
electrode was modiﬁed with Fe
nanoparticles; then the electropolymerization of neutral red in
ethaline solution was performed, followed by immobilization of acetylcholinesterase (AChE).
Kumar-Krishnan et al. demonstrated a simple and green synthetic approach based
on the use of reline for the amine-surface functionalization of silicon dioxide and the
integration of gold nanoparticles (AuNPs) for the preparation of a glucose biosensor [
the FAD-center of glucose oxidase (GOx) was covalently linked to the amine groups of the
NPs using glutaraldehyde as bifunctional cross-linker (Figure 8).
Thanks to the higher viscosity of reline in comparison to aqueous solvents, it was possible
to obtain uniform surface functionalization with better stability and dispersion.
Scheme of the amine functionalization of SiO
NPs (green) in DES, and the following immobilization of AuNPs
(yellow) and of glucose oxide (GOx) on the nanoparticle surface. (Adapted from  with permission).
Sensors 2021,21, 4263 13 of 18
Afterwards, GCEs were modiﬁed with Au-SiO
NPs/GOx and employed for glucose
quantitation. The biosensor showed direct electron transfer for the sensing of glucose with
a sensitivity of 9.69
, wide linear range from 0.2 to 7 mM, and very good stability.
This green DES-based synthetic procedure opens up the possibility of supporting different
metal nanoparticles on SiO
and, therefore, their subsequent use in the development
Two forms of nanocellulose obtained by exploiting a DES were applied by Ling
et al. as platforms to enable the colorimetric detection of human neutrophil elastase
]. A DES based on ChCl and oxalic acid was employed for the formation
of cotton cellulose nanocrystals (DCNCs), which were later used for the assembly of a
protease sensor for HNE based on a peptide–cellulose conjugate. A comparison between
the tetrapeptide–cellulose analog on DCNC and the analogous derivative of TEMPO-
oxidized wood cellulose nanoﬁbrils (WCNFs) was carried out. DCNCs proved to have a
higher degree of substitution of HNE tetrapeptide and also a better sensitivity compared
to WCNFs. X-ray diffraction (XRD) models showed the higher crystallinity and larger
crystallite sizes of DCNCs. The sensitivity of this colorimetric biosensor sensor was less
than 0.005 U/mL, reaching HNE levels that are usually found in human inﬂammatory
diseases. Moreover, the authors stated that the cellulose derived from this innovative
DES-mediated treatment could supply a new approach for the control of the nanocellulose
crystal sizes, content of carboxyl groups, and surface area, which usually are decisive
parameters for obtaining high-performing HNE sensors.
Svigelj et al. proposed a sandwich biosensor based on a truncated aptamer capable
of recognizing gliadin directly in a DES [
]. The sensor used Gli4-T as the capture and
signaling aptamer, one immobilized on the carbon working electrode and the other used
to reveal the binding of the target protein after incubation with streptavidin–horseradish
peroxidase (Figure 9). The biosensor showed a dynamic range between 1 and 100
and an intra-assay coefﬁcient of variation of 11%. With this analytical performance it was
possible to quantify 20
g of gluten per kilogram of food when 1 g of food was extracted
with 10 mL of ethaline. This aptamer-based sensor was directly deployed in ethaline, which
was used for the extraction of gluten proteins from food. The optimization of the aptamer
by truncation is a key step in the design. This sensor provides a new and faster way of
analyzing residual levels of gluten in food, after extraction in DES, offering a promising
tool to monitor and assess gluten in foods for highly sensitive celiac people.
Finally, Chen et al. described a composite generated by the coupling of a DES and
]. This composite was shown to be able to oxidize
-tetramethylbenzidine (TMB) to the oxidized blue product (oxTMB) which presents
a maximum absorbance peak at 652 nm. The DNA molecules, which are typically nega-
tively charged, were immobilized on the surface of the composite by hydrogen bonding
interactions and electrostatic interactions, thanks to the presence of DES. The adsorption
of DNA molecules caused the suppression of the oxidase-like activity of DES/MnO
The authors took advantage of this result to set up a colorimetric approach for
the detection of DNA. The DES-based system showed high selectivity for DNA against
several targets of biological nature; however, the presence of RNA in the sample can cause
interference. The sensor was also applied in the analysis of real samples, such as bovine
whole blood, demonstrating that this method could be easily used for the detection of DNA
in complex matrices. This assay has a dynamic range of 10–100
and a detection
limit of 0.37 µg mL−1.
Sensors 2021,21, 4263 14 of 18
Sandwich biosensor based on a truncated aptamer capable of recognizing gliadin directly in a DES: (
) Nyquist plots
of 0.5 mM [Fe(CN)
after each modification step involved in the assembly of the biosensor, which are schematically
represented in (
) Voltammetric measurements recorded in KCl 0.1 M with 0.5 mM [Fe(CN)
after each modification
) Steps involved in the sandwich assay; D1: interaction with the sample containing gliadin, D2: incubation with the
second aptamer labelled with biotin, D3: incubation with streptavidin–HRP conjugate and chronoamperometric measurement
of the oxidized tetramethylbenzidine enzymatically produced. (Reprinted with permission from ).
Ethanol, 90°C, 30 min
TMB = 3,3′,5,5′-Tetramethylbenzidine
Preparation of DES/MnO
and its application for the colorimetric detection of DNA. Adapted with permission
Sensors 2021,21, 4263 15 of 18
5. Conclusions and Future Perspectives
In this review, we collected recent progress in the use of DESs for the development of
biosensors. Considering their high compatibility with DNA, enzymes, and MIPs, which
are popular recognition elements, and their ability to stabilize nanomaterials, it is clear that
the use of DESs can bring relevant advantages for electrode modiﬁcations; furthermore,
these solvents can be exploited to develop biosensors with improved sensitivity and to
enable poorly soluble molecules to be detected.
Finally, the use of DESs could allow enormous advances in the ﬁeld of biotechnology:
they could play a signiﬁcant role in advancements in DNA-related technologies, enzyme-
based procedures, and MIP production. In the light of all DESs’ features, including their
easy preparation, biodegradability, high stability, and low volatility, the possibility of using
DESs in biosensing implementations appears to be of unquestionable interest. Despite the
great progress obtained in the last years, there are still many unexplored research routes in
biosensing development using DESs.
In particular, it is necessary to increase the knowledge about the inﬂuence of DESs
on the mechanisms of enzyme functioning and the interactions between aptamers and
ligands; also, the strategies of electrode material modiﬁcation with DESs should be further
expanded to lead to high-level technological developments.
Moreover, DESs’ ability to cover a wide range of polarity opens up the possibility of
creating analytical platforms for a series of molecules for which it has not been possible
to develop an effective methodology so far because of solubility reasons. For instance,
lately, DESs have been used as extraction solvents during the pre-concentration of pesticide
residues in food and environmental samples, with subsequent instrumental analysis .
Considering also the biocompatibility of these media with cells, DNA, proteins, and
enzymes, it is reasonable to think that they could play a relevant role in biosensing devel-
opments for biomedicine targets.
Finally, DESs are nontoxic and environmentally friendly, characteristics that make
them appealing for applications as green media in the development of devices for the
pharmaceutical, agrochemical, and food industries.
Conceptualization, R.S.; writing—original draft preparation, R.S. and R.T.;
writing—review and editing, N.D. and C.G.; visualization, R.S. and C.G.; supervision, R.T. and N.D.;
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Financial support from the Italian Ministry of University and Scientiﬁc Research
is gratefully acknowledged.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
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