Pnictogen-Based Enzymatic Phenol Biosensors: Phosphorene, Arsenene, Antimonene, and Bismuthene

Article abstract of DOI:10.1002/anie.201808846

Two-dimensional materials have allowed for great advances in the biosensors field and to obtain sophisticated, smart, and miniaturized devices. In this work, we optimized a highly sensitive and selective phenol biosensor using 2D pnictogens (phosphorene, arsenene, antimonene, and bismuthene) as sensing platforms. Exfoliated pnictogen were obtained by the shear-force method, undergoing delamination and downsizing to thin nanosheets. Interestingly, compared with the other tested elements, antimonene exhibited the highest degree of exfoliation and the lowest oxidation-to-bulk ratio, to which we attribute its enhanced performance in the phenol biosensor system reported here. The proposed design represents the first biosensor approach developed using exfoliated pnictogens beyond phosphorene.

Full text of DOI:10.1002/anie.201808846

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Accepted Article
Title: Pnictogens Based Biosensor: Phosphorene, Arsenene,
Antimonene, and Bismuthene
Authors: Martin Pumera
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201808846
Angew. Chem. 10.1002/ange.201808846
Link to VoR: http://dx.doi.org/10.1002/anie.201808846
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10.1002/anie.201808846
Angewandte Chemie International Edition
COMMUNICATION
Pnictogens Based Biosensor: Phosphorene, Arsenene,
Antimonene, and Bismuthene
Dedicated to memory of Prof. Emil Paleček, founder of electrochemistry of DNA
Carmen C. Mayorga-Martinez,[a] Rui Gusmão,[a] Zdeněk Sofer,[a] and Martin Pumera,*[a]
Abstract: Two dimensional materials have allowed for great
advances in the biosensors field and to obtain sophisticated, smart,
and miniaturized devices. In this work, we optimized a highly sensitive
and selective phenol biosensor using 2D pnictogens (phosphorene,
arsenene, antimonene and bismuthene) as sensing platforms.
Exfoliated pnictogen were obtained by the shear force method,
undorgoing delamination and downsizing to thin nanosheets.
Interestingly, compared with the other tested elements, antimonene
exhibited the highest degree of exfoliation and the lowest oxidation-
to-bulk ratio, to which we attribute its enhanced performance in the
phenol biosensor system reported here. The proposed design
represents the first biosensor approach developed using exfoliated
pnictogens beyond phosphorene.
analytical performance in term of sensitivity, selectivity, and
reproducibility. Moreover, to the best of our understanding, it is
the first example of a biosensing application reported for
pnictogens beyond phosphorene.
A promising application for novel 2D pnictogens in an enzymatic
biosensor was evaluate. Shear exfoliation method was used to
obtain the pnictogens nanosheets (Scheme 1A). The biosensor
device was constructed using a layer-by-layer method of each
pnictogen
(phosphorene,
arsenene,
antimonene,
and
bismuthene), enzyme (tyrosinase), and crosslinking agent
(glutaraldehyde, Glu) (Scheme 1B). The obtained biosensor was
used for phenol detection following the o-quinone
electroreduction to catechol (Scheme 1C). Tyrosinase (Tyr) is the
enzyme responsible for the two oxidation steps of phenol to
catechol and catechol to o-quinone.
Nowadays, the physical, chemical, electrical, and electrochemical
properties of two-dimensional (2D) layered materials are intensely
studied in the search for advanced applications in the
nanoelectronics, energy, biomedical, and environmental fields.[1,2]
Particularly for biomedical and environmental applications,
researchers in the development of smart and sensitive optical and
electrochemical biosensors have made tremendous gains
recently[3–6] with respect to precious nanoparticles.[7] The most
studied 2D materials for use as platforms or to label biosensor
applications are graphene, metal transitional dichalcogenides
(TMDs), and black phosphorus (BP), which demonstrate their
promising capacity in the development of this field of research.[6,8–
12]
Following explosive interest in the study of phosphorene’s
physicochemical properties and applications.[9,13–16] current
research focuses on other elements that adopt a layered structure
such as arsenene, antimonene, and bismuthene. These Group
VA elements are called “pnictogens” or “pnictides” (IUPAC
recommended designation).[17–22] The heavier pnictogens have
the advantage of a higher stability than phosphorene.[23] These
pnictogens beyond BP are not much explored and scientific
reports of their potential applications are currently limited,
particularly in the field of biosensing. Recently, scalable and
simple methods based on shear force exfoliation were developed
by our group and the obtained exfoliated pnictogens show high
electrochemical activity.[21-23] However, we believe that it is
necessary to explore other applications of these materials such
as biosensing. Herein, we optimized and implemented a phenol
enzymatic biosensor where exfoliated pnictogens (phosphorene,
arsenene, antimonene, and bismuthene) are used as a platform
for enhanced electron transfer. Interestingly, antimonene shows
the best performance and the resulting biosensor revealed high
Scheme 1. (A) Shear exfoliated pnictogens using kitchen blender. (B)
Biosensor preparation using layer-by-layer drop-casted pnictogen nanosheets,
tyrosinase (Tyr), and glutaraldehyde (Glu) onto a glassy carbon (GC) electrode.
(C) Chemical mechanism of phenol detection by biosensor based on exfoliated
pnictogen and Tyr.
Once the layered pnictogens (P, As, Sb, Bi) were exfoliated, a
structural and chemical characterization of their chemical
composition and morphology was performed. Transmission
electron microscopy (TEM) images (Figure 1: left and central
panels) clearly show the extent of exfoliation and the lateral size
of the exfoliated pnictogens. Interestingly, each material shows a
different degree of exfoliation. Antimonene and phosphorene
show the typical 2D structure of a few layers (Figure 1A and 1D).
Antimonene displays well-defined nanosheets with lateral size of
around 200 nm and underwent a substantial downsizing from the
bulk. Large sheets of arsenene with a wrinkled structure were
observed (Figure 1B) and agglomerated nanoparticles of
bismuthene (Figure 1C) were obtained. Moreover, the selective
area electron diffraction (SAED) patterns and HR-TEM images
show the crystallinity corresponding to the rhombohedral (As, Sb,
and Bi) and orthorhombic (BP) structures of the exfoliated
pnictogens (see Figure 1: right panels and inset). The EDX
[a]
Dr. C.C. Mayorga-Martinez, Dr. R. Gusmão, Prof. Dr. Z.
Sofer, Prof. Dr. M. Pumera*
Center for Advanced Functional Nanorobots, Department of
Inorganic Chemistry, University of Chemistry and
Technology Prague, Technicka 5, 166 28 Prague 6, Czech
Republic
E-mail: martin.pumera@vscht.cz
Supporting information for this article is given via a link at the
end of the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201808846
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COMMUNICATION
mapping from the TEM images (see Figure S1) confirmed the
presence of the element for each pnictogen and the oxygen
content. The bulk materials were also investigated by X-ray
diffraction (spectra provided in Figure S2). All samples were
single-phase corresponding to orthorhombic black phosphorus
and rhombohedral arsenic, antimony, and bismuth. Within the
arsenene was observed arsenic oxide due to partial oxidation,
which means that the crystalline structure remains intact after the
exfoliation process. The surface composition, chemical bonding
of the bulk, and exfoliated pnictogens were evaluated by X-ray
photoelectron spectroscopy (XPS) (see Figure S3 in SI). The
exfoliated materials displayed a partial oxidation from the starting
materials, but it is also interesting to mention that in the case of
antimonene, its bulk shows a significant degree of oxidation in
comparison with the other bulk materials (Bi, As, and BP). In this
manner, the oxidation ratio of the antimonene is the lowest of the
series.[23] After the exfoliated pnictogens were properly
characterized, we proceeded to evaluate their applications as
nanocarriers to enhance the activity of an enzyme-based phenol
biosensor. In this manner, we fabricated and optimized the
biosensing system using exfoliated pnictogens. First, we
prepared different biosensors using each pnictogen and their
electrocatalytic responses were measured by cyclic voltammetry
(Figure 2A). The cyclic voltammograms (CVs) were carried out in
phenol 200 µM solution prepared in PBS pH 6.5. The catalytic
activity of this phenol biosensor based on tyrosinase (Tyr) was
electrochemically detected by the evaluation of a reduction peak
corresponding to the electroreduction of o-quinone to catechol at
negative low potentials (Scheme 1C). Particularly, in the phenol
biosensor described here, the o-quinone reduction peak occurs at
around −43 mV for antimonene, −63 mV for phosphorene, −30
mV for bismuthene, and −4 mV for arsenene; however, current
intensity occurs in the following order: antimonene > phosphorene
> bismuthene > arsenene. The electrochemical mechanism of this
biosensor system is based on three successive reactions. First,
phenol hydroxylation to catechol in presence of tyrosine (reaction
1) followed by the catechol oxidation to o-quinone by tyrosinase
(reaction 2) and, finally, the electro-reduction of o-quinone to
catechol (reaction 3).[24] Remarkably, all the exfoliated materials
show some reduction peak of o-quinone; however, antimonene
(red curve) shows the best performance based in its current
intensity. The role of each layer of the antimonene-base biosensor
(Figure S4 A) and its response at different phenol concentrations
Figure 1. Transmission electron microscopy at different magnifications (left and
central panels), high-resolution TEM (right panels), and selective area
diffraction (insets) of antimonene (A), arsenene (B), bismuthene (C), and
phosphorene(D) shear-exfoliated nanosheets.
Comparison with biosensors prepared using Sb bulk (red dash
line) and glassy carbon electrode (black dash line) (Figure S5)
shows the enhanced activity of the antimonene based biosensor
for phenol detection. Moreover, similar signals for bulk layered Sb
and GC were observed, which means that the enzyme activity is
not inhibited by neither bulk nor exfoliated antimony. Bismuthene
and phosphorene yielded signals similar to the control
measurements (biosensor without pnictogens, Figure 3A), which
demonstrates that although they do not inhibit their enzymatic
activity, these materials have not improved the biosensor
electroactivity. In order to elucidate the role of the antimonene in
the observed enhanced signal in this biosensing system, different
experiments were performed. First, the ability of the exfoliated
pnictogens for o-quinone reduction to catechol was evaluated.
GC electrodes were modified with each pnictogen and the
electrochemical process of catechol oxidation to o-quinone[30,31]
was evaluated, see Figure 2B. A single oxidation peak of catechol
oxidation to o-quinone was observed around +0.35 V and the
peak corresponding to o-quinone reduction was observed at
+0.11 V during reversed scan. However, arsenene shows
enhanced catechol oxidation (green line), whereas antimonene
reduced the o-quinone to catechol more efficiently (red line).
Secondly, the electroactivity of phenol in presence of pnictogens
was also evaluated (Figure 2C). In this case, only an irreversible
phenol oxidation peak at higher anodic potentials was observed
for all pnictogens ca. +0.7 V, thus demonstrating that the
exfoliated pnictogens are not able to detect phenol without the
presence of Tyr.
(Figure S4 B) were evaluated (see Supporting Information) as well.
푇푦푟
푝ℎ푒푛표푙 퐶6퐻5푂퐻 + 퐻2푂 ⇒ 푐푎푡푒푐ℎ표푙 (퐶6퐻6푂2) + 2퐻++ 2푒−
푐푎푡푒푐ℎ표푙 (퐶6퐻6푂2) ⇒푇푦푟 표 −푞푢ꢀ푛표푛푒(퐶 퐻 푂 ) + 2퐻++ 2푒−
(1)
(2)
(3)
(
)
−50 푚푉
푎푛푡푖푚표푛푒푛푒
6
4 2
(
)
표 −푞푢ꢀ푛표푛푒 (퐶6퐻4푂2) + 2퐻++ 2푒−
⇔
푐푎푡푒푐ℎ표푙 (퐶6퐻6푂2)
It is of importance to mention that the pnictogens show thiophilic
characteristics, binding to the cysteine group of the proteins.[30]
This is the reason that the enzyme molecules are well immobilized
onto the pnictogens. It is well know that arsenic compounds are
able to inhibit up to 200 enzymes[26-28] Herein, when the arsenene
was used as platform for this tyrosinase-based biosensor, the
signal is lower (green line in Figure 2A) in comparison to the
carbon biosensing platform (black dash line in Figure 2A). This is
likely due to enzyme inhibition by arsenene. In contrary,
antimonene shows enhanced signal (red line), which confirms
that this material does not inhibit the enzyme activity.[29]
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10.1002/anie.201808846
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Figure 2. (A) CVs of biosensors based on phosphorene, arsenene, antimonene, and bismuthene for phenol (200 µM) detection. Electrocatalytic activity of the
exfoliated pnictogens in presence of (B) catechol 200 µM and (C) phenol 200 µM. (D) Cathodic scan of the antimonene based biosensors in presence of phenol
200 µM. Experimental conditions: 0.1 M PBS pH 6.5, and scan rate 50 mV/s.
The enhanced performance of the antimonene-based phenol
biosensor can be assigned to three factors: (i) a degree of
exfoliation and downsizing as well-observed in the TEM images
(Figure 1A), (ii) the lowest ratio of oxidation of its surface observed
in the XPS studies (Figure S3) and (iii) the biocompatibility of
antimonene (Figure 2A). These findings reveal antimonene with
enhanced electron transfer capability suitable for biosensors
application. Consequently, antimonene was the chosen exfoliated
pnictogen for the fabrication of the phenol biosensor.
In a third sets of experiments, considering that the phenol
detection in this biosensor system is based on o-quinone
reduction, antimonene based biosensor was prepared and
evaluated. Figure 2D shows cathodic voltammetric scan, from
+0.2 to -0.4 V in presence of 200 µM of phenol. The antimonene-
based biosensor shows a cathodic peak at -0.01 V that
corresponds to o-quinone reduction. This confirms that the o-
quinone production and subsequent reduction by the antimonene-
based biosensor is not dependent on any electro-oxidation pre-
process. In contrast, anodic potentials oxidize phenol, thus
competing with the enzyme for the substrate and decreasing the
performance of the pnictogenene-based biosensor. CV was
measured at different scan rates from 5 to 400 mV/s at the
antimonene-based biosensor in presence of phenol 200 µM to
have an insight whether the process is diffusion or adsorption
controlled (Figure S6). The reduction peak current (reaction 3) is
proportional to the scan rate, which mean that this is an
adsorption controlled electrochemical process.
We optimized the detection potential in wide range of potential
(Figure S4 C in Supporting Information). Subsequently, once the
most favorable working potential was optimized (−5 mV) the
chronoamperogram of various phenol concentrations was
recorded (Figure 3). Remarkably, we observed the current drop
as a small step in the presence of 500 nM of phenol,
demonstrating the high sensitivity of the biosensor implemented
and electrochemical detection method used as well. This
enzymatic biosensor system operates following the Michaelis-
Menten kinetics scheme, see the current response vs.
concentration plot (Figure S7 A). Calibration curves from
chronoamperograms are shown in Figures S7 B and C.
Calibration and analytical data are stated in the Supporting
Information.
The
phenol
biosensor
based
on
antimonene
(antimonene/Tyr/Glu) showed good analytical performance with
high sensitivity, selectivity, reproducibility, and specificity. Taking
in account the high toxicity of phenolic compounds, which have
an exposure limit of 5 µM in drinking water,[32] this biosensor
represents a good alternative for phenolic compound detection in
environmental control applications as its LOD is 10 times lower
than this limit. The influence of interferences (Figure S8 A) and
the recoveries of spiked tap water (Figure S8 B and C) for its
future application in real samples are discussed in Supporting
Information. The sensitivity of this biosensor is comparable with
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and bismuthene) obtained by shear force exfoliation as a platform
for the detection of phenol. Exfoliated pnictogens undergo
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antimonene exhibited the highest degree of exfoliation and the
lowest oxidation-to-bulk ratio. Simultaneously, the phenol
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group VA elements. The improved electrocatalytic reduction of
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Research was supported by the project Advanced Functional
Nanorobots (reg. No. CZ.02.1.01/0.0/0.0/15_003/0000444
financed by the EFRR). Z.S. is supported by the Czech Science
Foundation (GACR No. 16-05167S). This work was created with
the financial support of the Neuron Foundation for science support
(Z.S.).
Keywords: Pnictogen, phosphorene, monoelemental layered
material, electrochemical biosensor, toxic compound, phenol
biosensor
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COMMUNICATION
Keyword: Biosensing
Carmen C. Mayorga-
Martinez, Rui Gusmão,
Zdeněk Sofer, and Martin
Pumera
Page No. – Page No.
Pnictogens Based
Biosensor:
Phosphorene,
Arsenene, Antimonene,
and Bismuthene
Highly sensitive and selective phenol biosensor using 2D antimonene as a platform.
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