j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5

Available online at www.sciencedirect.com

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

Original Article

Solution grown multilayered zinc vanadium oxide

nanosheets as a new material to bind glutathione

Ayesha Taj a,b, Hafsa Maqsood c, Rabisa Zia a,b, Adnan Mujahid d,
Adeel Afzal d, Zeeshan Tariq e, Faheem K. Butt c,***, Waheed S. Khan a,**,
Sadia Z. Bajwa a,*
a
Nanobiotechnology Group, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road,
Faisalabad 38000, Pakistan
b
Pakistan Institute of Engineering and Applied Sciences, Nilore, Islamabad 45650, Pakistan
c
Department of Physics, Division of Science and Technology, University of Education Lahore, 54770, Pakistan
d
School of Chemistry, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan
e
School of Science, Minzu University of China, 100081 Beijing, China

article info abstract

Article history: Development of layered nanostructures with distinct morphology has become an avenue of
Received 18 July 2020 recent research to engineer targeted properties, especially for electrochemical devices. How-
Accepted 19 January 2022 ever, their remarkable characteristics are yet to be exploited for biological applications. In this
Available online 29 January 2022 work, we followed a facile and single-step method and prepared the solution grown zinc va-
nadium oxide multilayered nanosheets (ZnV2O4 MLNs). Field emission scanning electron
Keywords: microscopy shows the presence of nanosheets about 15e20 nm in height, integrated, and
Zinc vanadium oxide overlapped with one another, conferring a layered morphology. The layers are a structural
Multilayer nanosheets mixture of triangular and hexagonal shapes. The phase pure and highly crystalline ZnV2O4
Solution grown synthesis MLNs are investigated as an efficient receptor material for the electrocatalytic binding of
Electrochemical biosensor glutathione, an important biomolecule involved in many cellular processes. This work is the
Biomolecules first report where ZnV2O4 MLNs are employed to bind biomolecules. These nanostructured
interfaces are expected to increase the effective surface area for detection, improve the planer
surface of the sensor, and provide more binding sides for greater sensitivity. The developed
sensor enables the sensitive detection of glutathione with outstanding detection limit down to
9 nM and ensures the 100% selectivity at a specific potential when compared to its functional
and structural analogues. The study demonstrates the novel multilayer morphology-based
interfaces can be a favourable platform to develop immune probes and further establishes
their great potential to detect clinical biomolecules and disease biomarkers.
© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: faheemk.butt@ue.edu.pk (F.K. Butt), waheedskhan@yahoo.com (W.S. Khan), sadya2002pk@yahoo.co.uk, sadia.
zafar.bajwa@nibge.org (S.Z. Bajwa).
https://doi.org/10.1016/j.jmrt.2022.01.110
2238-7854/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
1778 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5

high technical inputs. To meet these challenges, electro-

1. Introduction chemical based methods can be preferred due to their
inherent simplicity, low background noise, sensitivity, broad
Layered materials are receiving great attention due to their dynamic response range and rapidity, to name a few [21].
outstanding structural, electrical, and mechanical properties The development of biosensors capable of detecting low
[1]. They possess unique optical band gap, quantum confine- concentrations of analytes has been the focus of clinical di-
ment, and large specific surface area [2]. Among layered ma- agnostics, food safety and environmental monitoring [13]. The
terials, multilayer nanostructures are the new focus of most essential requirement is creating optimal sensing sur-
research to increase the efficiency of other applications such faces or transducers that can efficiently interact with the an-
as catalysis, spintronics, solar energy conversion etc. [3]. The alyte molecules to transform biorecognition into a measurable
versatility of layered materials can be used sometimes as- signal [16]. Therefore, selecting an appropriate sensing ma-
prepared and/or combining with other functionalities [4]. terial possessing recognition and transduction capabilities is
Such layered materials, with tailored properties can be a very critical in obtaining the required performance. The recent
promising candidate, especially for the catalysis [3]. However, advancement of layered and precisely constructed hierarchi-
the synthesis of multilayer nanostructure is a critical issue in cal nanostructures opens the door to the development of
developing a useful application. To fabricate their patterned innovative transducers. The layered materials provide me-
layers, traditional methods, such as lithography entail limi- chanical stability, the quantum size effect as well as essential
tations and relatively high cost [5]. Therefore, facile, single- surface characteristics [4]. Due to their outstanding proper-
step, and cost-effective methods are highly required. The ties, these materials can be attractive choices for the selective
size, morphology, and chemical composition have a signifi- and sensitive detection of analyte for diagnostic applications.
cant impact on the performance and utility of nanomaterials In this work, a simple material structuring technique is
[6]. Different chemical composition and morphology of the adopted to synthesize the zinc vanadium oxide multilayer
layered materials can be investigated to develop beneficial nanosheets and further they are integrated with an electro-
utilization, especially in the biological and biomedical chemical platform to design a sensor interface. This is the first
applications. report describing a facile route to synthesize multilayer
Spinels are a very specific type of layered materials [7]. nanosheets and unveiling its binding properties and unique
They have an isometric crystal structure with a general for- morphology to recognize glutathione. The fabricated interface
mula of AB2X4, here A and B can be different or same metal exhibits outstanding electrochemical signals for the selective
ions with different valences [7,8]. Few metals like Cr3þ, Fe2þ detection of glutathione. This study opens new pathways to
and Zn2þ react with other metal ions to form spinel with design such multilayered nanomaterials for manufacturing
assorted symmetrical patterns [9]. The crystal structure of multipurpose applications, and demonstrate the promising
normal spinels is cubic closed packed oxides. The cubic potential of such materials in diagnostics.
morphology of spinels consists of eight tetrahedral sites and
four octahedral sites. Spinels have gained more attention due
to their structural flexibility, stability and a minimum of two 2. Experiments and methods
orders of magnitude, higher electrical conductivity than the
equivalent single-metal oxide [10]. Spinel-based materials 2.1. Preparation of ZnV2O4 MLNs
exhibit better catalysis because of their controllable compo-
sition, structure, valence, and morphology [11]. Zinc and va- In a typical synthesis, zinc acetate dehydrate (0.5 g) was added
nadium both are earth abundant elements. They are in 15 mL of distilled H2O and the beaker was kept on gentle
affordable with excellent magnetic and catalytic activity [12]. stirring at room temperature. Afterwards, ammonium meta-
The cubic ZnV2O4 spinel nanostructures consist of eight vanadate (0.24 g) was added with vigorous stirring until a ho-
tetrahedral sites and four octahedral sites. They show mogenous mixture was obtained. Then, oxalic acid (0.5 g) was
different oxidation states that give a suitable micro- added as a chelating agent. This was followed by the addition of
environment for the electrochemical catalysis of glutathione nitric acid (15.8 M; 70%) and hydrogen peroxide (30% w/w in
by a redox reaction [13,14]. H2O) in equal volumes (4 mL, each) directly in the reaction
Glutathione is a most prevalent tripeptide (l-Y-glutamyl-L- mixture. The final solution was then poured into an autoclave
cysteinylglycine) known as intracellular thiol. It is involved, (50 mL) with Teflon lining and placed in the oven for 24 h at 200
directly or indirectly, in many biological processes like syn- 
C. After 24 h it was taken out and allowed to cool naturally at
thesis of DNA, transportation of protein and enzyme activity ambient temperature (25  C). A powder of black color was ob-
[15]. It is an important naturally occurring antioxidant found tained and this was rinsed three times with absolute ethanol.
in the human body which neutralizes the potentially toxic Eventually, the product was vacuum dried at 80  C for 8 h and
compounds. Moreover, it acts as a biomarker to indicate the stored in the desiccator. The same was further annealed at 300
important biochemical changes occurring in the body as the 
C for 2 hrs. For all experiments and preparation of the solu-
result of an ailment [16,17]. There are some conventional tions, ultrapure water (r ¼ 18 MU cm) was used.
techniques which are widely used to determine the presence
of this biomolecule, such as high-performance liquid chro- 2.2. Characterization and instruments
matography [18], enzymatic based assay [19], and capillary
electrophoresis [20]. They methods involve some inherent To obtain the morphology and structural features of ZnV2O4
drawbacks such as high cost, time consumption, and above all MLNs, field emission scanning electron microscopy (FESEM
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5 1779

Fig. 1 e Microscopic illustration of ZnV2O4 MLNs. (AeB) FESEM image of ZnV2O4 MLNs at different magnification 5 mm and
1 mm (C) AFM topograph of ZnV2O4 MLNs with individual nanosheet section analysis.

Hitachi S-4800) and atomic force microscopy (AFM SHI- electrodes were further washed with nitric acid followed by
MADDZU WET-SPM 9600 Japan) were used. For these experi- rinsing with ethanol. Finally, intensive cleaning was carried out
ments, samples were prepared by drop coating 10 mL dispersion with distilled water. After drying with the stream of nitrogen
of ZnV2O4 (1 mg mL1) on 0.5 cm2 glass slides. To obtain AFM gas (99%), 10 mL of ZnV2O4 MLNs (5 mg 10 mL1 suspension) was
topographs, a silicon nitride cantilever (model OMCL- dropwise coated on the shining side of glassy carbon electrodes
TR800PSA-1) was adjusted in the contact mode. Cantilever (GCEs). The subsequent layers of MLNs were protected with
has 100 mm thickness and force constant of 0.57 N m1. SPM 0.2 mL of Nafion (2%). Designated as a ZnV2O4 MLNs sensor, the
Manager Software was utilized for data analysis which was electrodes were dried at ambient temperature, overnight. The
provided by AFM system supplier. To get representative clean and dried bare GCEs were used as the control materials to
morphology, at least three different samples of the same ma- compare the efficiency of the ZnV2O4 MLNs sensors.
terial were scanned under the similar conditions. X'pert Pro
PAN analytical diffractometer was used to record X-ray 2.4. Electrochemical studies
diffraction (XRD) spectra of ZnV2O4 MLNs with the help of
monochromatic CuKa radiation (l ¼ 1.5406A) working at 40 kV A Potentiostat/Galvanostat (PGSTAT; Autolab, AUT72284,
and current of 30 mA. Raman spectrometer (Renishaw in Via Netherlands) having a three-electrode system was used to
System with a 633 nm laser) was used to record the Raman observe the catalysis of glutathione (electrochemical) by
spectra of ZnV2O4 MLNs using a laser source of 633 nm as an cyclic voltammetry (CV). The MLNs modified electrodes
excitation source. were used as the working electrodes. For reference and
counter electrodes, Ag/AgCl (3M KCl) and a glassy carbon
2.3. Fabrication of ZnV2O4 MLNs sensor interfaces rod were used, respectively to support the redox reactions
between sensor surface and glutathione. All measure-
To prepare the interfaces, glassy carbon electrodes were ments were carried out in background media of sodium
cleaned by different size grades of alumina slurry (1.0, 0.3, and phosphate buffer (50 mM). The free oxygen species of the
0.05 mm). To ensure complete surface availability, the media were removed by purging nitrogen gas (99%) about
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Fig. 2 e Structural analysis and sensor signal of bare and ZnV2O4 MLNs modified electrode, (A) XRD pattern of ZnV2O4 MLNs
{JCPDS card no. 65e3108}, (B) Crystal structure of FCC ZnV2O4 MLNs in which Zinc, vanadium, and oxygen atoms are presented
by blue, green, and red colour respectively, (C) Raman spectra of the intermediate product (Zn2(OH)3(VO)3) and (D) ZnV2O4 MLNs,
(E) CV responses of bare and ZnV2O4 MLNs modified GCE in 5 mM [Fe4 (CN)6] and 0.1 M KCl at a fixed scan rate of 100 mVs¡1.

for 5 min, before each experiment. Except for the inves- morphology of ZnV2O4 MLNs. Fig. 1A reveals that the material
tigation of its impact on sensor performance, all cyclic is composed of numerous and dense layers of ZnV2O4 nano-
voltammetry responses for glutathione binding were ob- sheets. They are integrated with one another and overlapped
tained at a sweep rate of 100 mVs1. The potential range to produce a composite structure of about 100e500 nm in size.
during the electrochemical investigation of glutathione Further, the FESEM image of higher magnification (Fig. 1B)
was selected between negative to positive 1.0 V, at suggests that the individual nanosheets bear different shapes,
25 ± 2  C. However, for the electrochemical character- such as triangular, hexagonal, etc. The thickness of a sub-
ization of the ZnV2O4 MLNs modified GCE, the CV curves stantial number of ZnV2O4 nanosheets was found to be
were acquired in the reaction media containing 5 mM [Fe4 10e20 nm as evidenced by the AFM micrograph (Fig. 1C). The
(CN)6] and 0.1 M KCl at a scan rate of 100 mVs1 and ultrathin morphology of nanosheets was assumed to confer
temperature of 25  C. remarkable binding characteristics.
The thickness of multilayer nanostructures was calculated
using SPM System software. Four different areas in AFM
3. Results and discussion micrograph were marked (SI. 1) and their profiles indicated
that the height or thickness of these multilayers varies from
3.1. Characterization of as-synthesized ZnV2O4 MLNs 10 to 50 nm. We selected a bundle of multilayer at a single
point and marked different cross sections to calculate the
The field emission scanning electron microscopy (FESEM) and thickness (SI. 2). The value of about 25e137 nm reveals that
atomic force microscopy (AFM) were used to investigate the layers possess varying thickness. This type of
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5 1781

Fig. 3 e Shows the electrochemical signals of designed sensor, (A) The electrochemical CV signals of bare glassy carbon
electrode and ZnV2O4 MLNs modified electrode towards 25 mM of glutathione; background electrolyte NaH2PO4. H2O (50 mM),
scan rate of 100 mVs¡1 and (B) CV responses of ZnV2O4 MNLs modified interface towards varying glutathione
concentrations (0.1e50 mM), inset shows the corresponding linear relation plot with background electrolyte NaH2PO4. H2O
(50 mM), scan rate of 100 mVs¡1.

multilayered materials with varying thickness have already ZnV2O4 MLNs peaks appeared at 258.4 cm1, 389.7 cm1,
been investigated by AFM [22e24], which further supports this 538 cm1, 838 cm1, 898 cm1 and 951 cm1 (Fig. 2D). However,
analysis. due to the increased number of peaks, obviously their position
Overall, The SEM and AFM analysis show that the as- and width change and they are slightly shifted towards higher
prepared structures bear multilayers which appear porous in wavelength. This may be ascribed to the reduced particle size
nature. Certainly, improved binding of analyte can be ex- and higher charge mobility obtained in case of ZnV2O4 MLNs.
pected, which in turn may lead to the enhanced electro- These observations are in accordance with the previously re-
catalysis. This morphology shows further special features: (i) ported results [12,29].
the multilayers, which can offer a larger surface area for The acquisition of cyclic voltammogram in the ferricyanide
interface reaction [25] (ii) the presence of various geometrical reaction system is considered a standard protocol to monitor
features can ensure optimal reaction centres [6] (iii) the the electrochemical characterization of modified electrodes
macroporous architecture of the layers can allow facile [20]. For this purpose, sensor responses of bare and ZnV2O4
diffusion of the analyte through multilayers to ensure elec- MLNs modified GCE were recorded in the reaction media
trocatalysis of the analyte [26]. containing 5 mM [Fe4 (CN)6] and 0.1 M KCl at 100 mVs1
To evaluate the possible reaction mechanism in the syn- (Fig. 2E).
thesis of multilayer ZnV2O4 nanosheets. The oxalic acid acts Cyclic voltammograms of bare (blue line) and modified
as a chelating agent and leads the gold yellow color of the glass carbon electrode (red line) are compared. The modified
solution that indicates the chelation of metal cations by oxa- electrode shows well-defined redox curves with high current
late anions, already reported in literature [27]. After that, the density (82 mA) at low potential (0.17 V), in contrast of bare
addition of HNO3 and H2O2 in the above solution gives electrode showing 38 mA at 0.27 V. The peak potential differ-
exothermic reaction by producing the HCOOH. The acid pro- ence of about 100 mV indicates the presence of more catalytic
duction in a solution leads to the reduction of vanadium giv- sites for the modified electrodes. In the case of multilayers, it
ing ZnV2O4 as a final product. can be assumed that complete interfacial contact between the
The structural properties of Zn MLNs were further inves- layers ensured seamless electron transport, which may have
tigated by X-ray diffraction (Fig. 2A). The strongest peaks (220), contributed to high current values [21]. Undoubtedly, these
(311), and (440) are observed at positions q ¼ 35.395, q ¼ 30.135 findings suggest superior electrochemical characteristics of
and q ¼ 62.492, respectively, that indicates its good crystalline ZnV2O4 MLNs, which can play an important role in the catal-
geometry with consistence Fd3m symmetry [13,28]. Some ysis of glutathione.
minor peaks can also be found including (400), (422), (511) and
(533) at different positions standardized with {JCPDS card no. 3.2. Detection of glutathione
65e3108}. The tetrahedral geometry of ZnO4 and octahedral of
VO6 shows that zinc vanadium oxide consists of a face centred The binding properties of synthesized ZnV2O4 MLNs were
cube (FCC) spinel structure. This structure has been depicted investigated by cyclic voltammetry technique. The electro-
in Fig. 2B. chemical responses were recorded for binding of glutathione.
Raman spectra can be found in Fig. 2 (CeD), which were Fig. 3A shows representative CVs of bare glassy carbon and
recorded to explain the vibrational aspects of different groups modified electrodes, towards 25 mM glutathione. In the case of
present in ZnV2O4 MLNs and its intermediate product. The bare GCE insignificant response was obtained (purple line
peaks were investigated from range 100 cm1 to 2000 cm1. curve) showing it is inefficient to support the transfer of
The characteristic sharp peaks of Zn2(OH)3(VO)3 were found at electrons on its interface. On the other hand, the modified
176.2 cm1 and 473.6 cm1 (Fig. 2C) while in the case of pure electrode with ZnV2O4 MLNs generated a prominent response
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Table 1 e Comparison of glutathione sensor performance.

Material Technique Linear Limit of detection Reference
range (LoD) nM
mM
Silver nanoparticles anchored reduced graphene oxide Amperometry 0.1e2.75 100 [30]
nanosheets
Ferrocene dicarboxylic acid modified carbon nanotubes Differential pulse voltammetry 0.5e24 200 [31]
paste electrode
AgeZnO nanoplates Square wave voltammetry 500e20000 20 [32]
Multi-wall carbon nanotubes/Platinum Hydrodynamic voltammetry with 0.2e100 29 [33]
liquid chromatography
Cobalt tetra-sulfonated phthalocyanine/Poly(L-lysine) Amperometry 0.5e3.5 80 [34]
Copper hydroxide composite carbon ionic liquid electrode Cyclic voltammetry 1e50 30 [35]
Pyrogallol red modified carbon nanotube paste electrode Square wave voltammetry 0.3e500 190 [36]
Cobalt phthalocyanine on a thioctic acid modified gold Differential pulse voltammetry 10e100 5500 [37]
electrode
Multi-wall carbon nanotubes/Isoprenalinene Sweep voltammetry 0.5e300 90 [38]
Zinc vanadium oxide multilayer nanosheets Cyclic voltammetry 0.1e50 9 This work

(red line curve) with a defined pair of redox peaks. The forward wide working capability for the binding of glutathione. Aw
oxidation peak current intensity of 142 ± 0.3 mA at lower linear relationship as found as depicted by the calibration
oxidation potential of 0.31 V demonstrates the improvement curve, between the concentration of glutathione and peak
of redox activity. The current in higher amount implies the current yielding satisfactory regression value (R2 ¼ 0.994).
ZnV2O4 MLNs bear conducting reaction sites supporting Other significant analytical parameters that meet the
electrocatalysis. Both anodic and cathodic peaks indicate a requirement of sensor for clinical applications include the
reversible redox reaction. The anodic (0.31 V) and cathodic limit of quantification (LoQ) and the limit of detection (LoD).
(0.27 V) peak potential separation was calculated to be about They were estimated to be 9 nM (S/N ¼ 3) and 29 nM (S/N ¼ 10),
40 mV indicating fast kinetics and greater capabilities of respectively using the formulae given below:
ZnV2O4 MLNs for the electrocatalysis of glutathione. These
results exhibit they can be excellent candidates to bind bio- LoD ¼ 3.3s/Slope
logical analytes and thus qualify for clinical diagnostics. There
are several reports in literature discussing the electrochemical LoQ ¼ 10s/Slope
investigation of glutathione by different metal nanostructures
including Zn nanosheets and bare GCE. The bare GCE was Where, the s stands for the standard deviation.
unable to initiate the catalysis of glutathione but the oxidation The high sensitivity of the designed material is accom-
potential by Zn nanostructures was found to be 0.28 V with the panied with the metallic nature and unique morphology of
lower detection value of 20 nM [30,32]. The value of oxidation highly populated and dense ZnV2O4 MLNs. These features
potential of glutathione is found in agreement as reported in enhance the conducting and catalytic activity. These results
the already published articles. This validates the GCE was confirm that along with the special morphological features,
successfully modified by our designed sensor material as the certainly the chemical composition of the nanostructures is
electro-catalytic performance of the interface. We attempted playing a pivotal role towards excellent electrocatalysis.
to investigate the underlying mechanism of the electro- This validates the designed interface can be applied suc-
chemical catalysis of glutathione (1). The oxidation of gluta- cessfully for the binding of glutathione. This is the first
thione involves the nucleophilic reaction giving the unstable study describing the potential of multi-layered structures.
specie (GS). These two unstable species interact by disulfide Nonetheless, we have compared our results with other
bond and covert into a stable product (GSSG) by giving 2 analytical techniques (Table 1). It can be observed that LoD
numbers of electrons [13]. These electrons react with the of ZnV2O4 MLNs is remarkable as compared to other mate-
multilayer ZnV2O4 sensing surface generating both cathodic rials used in the analytical detection of glutathione. The
and anodic peaks as an electroanalytical signal in outstanding morphology of ZnV2O4 MLNs with higher sur-
electrochemistry. face area provides more electronic cloud that enhances the
flow of electron for the electrochemical analysis. The sensor
k1
2GSH % GSSG þ 2e þ 2Hþ (1) response depends on the availability of active sites, the
k2
populated and layered nanostructure offer an enhanced
The analytical characteristics of fabricated sensors were number of active sites so that the analyte can diffuse
studied (Fig. 3B) by challenging the ZnV2O4 MLNs modified through it.
electrodes towards the glutathione (0.1e50 mM). The current of The kinetics were studied by observing the variation in
anode and cathode peak increases from 12.2 ± 0.1 mA to anodic and cathodic peak current, by varying the rate of scan
142 ± 0.2 mA and 8.2 ± 0.1 mA to 75 ± 0.2 mA, respectively, as the from 0.1 to 1 Vs1 towards 25 mM glutathione. The results are
concentration of analyte increases. The sensor shows the shown in Fig. 4A. It is seen that reduction and oxidation peak
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5 1783

Fig. 4 e CV curves showing the scan rate of ZnV2O4 MLNs. (A) CV curves showing the variation of scan rate of ZnV2O4 MLNs
modified interface 25 mM glutathione, and Experimental conditions are with background electrolyte NaH2PO4. H2O
(50 mM) (B) The corresponding linear regression relation plot in scan range 0.1e1 Vs-1.

current increase as the scan rate increases indicating an effi- interfering types of chemical analogues with analytical accu-
cient charge transfer kinetics process of ZnV2O4 MLNs. A linear racy. Further, the oxidation potentials in case of competing
relationship can be found between the square root of scan rate analytes were found different. This validates the designed
and current (co-relation coefficient 0.99; inset of Fig. 4B) ZnV2O4 sensor can differentiate biomolecules. This evidence
implying the electrocatalysis redox reaction of glutathione by indicates its potential for the selective recognition of
ZnV2O4 MLNs@ GCE is a diffusion-controlled process. glutathione.

3.3. Interference studies 3.4. Repeatability and stability

The major molecules that can interfere with the binding ac- Regeneration of sensor response is a significant factor deter-
tivity of glutathione can be lysine, glycine, tyrosine, leucine, mining the sensor's practical applications. Five different
glucose, histidine, and cysteine. They are structurally related layers of the same quantity of ZnV2O4 MLNs were investigated
compounds and co-exist in the biological systems. Therefore, by depositing over the electrode and exposed to 50 mM gluta-
we investigated electrochemical responses of these competi- thione, separately. The CV responses were obtained by keep-
tors in addition to glutathione (50 mM each) to assess the ing the other conditions of experiment constant, whereas
selectivity of our designed interface, keeping all other condi- every single response of the sensor is determined as the
tions same. The results are presented in Fig. 5. The modified average of ten consecutive scans. Summarized results are
electrodes show outstanding electrochemical responses to- displayed in Fig. 6A which shows the stability of electro-
wards glutathione yielding the peak current intensity of chemical signals. Additionally, around 99% signals are repro-
142 mA. Conversely, the responses of interfering agents are low ducible which shows the analytical potential of fabricated
(Table 2) showing that the structures can distinguish these sensors. This strongly suggests the suitability of the designed
sensor to be used in real life applications.
Moreover, by recoding CVs towards 50 mM glutathione, the
stability of the fabricated sensor was studied for 5 successive
days at 100 mVs1 scan rates for the same modified electrodes.
The results are depicted in Fig. 6B. Statistically the values of
current are same, demonstrating the analytical stability of the
ZnV2O4 MLNs interface. These results ensure high coverage of
surface by the structures and therefore providing greater
possibilities of the binding sites which might have contributed
towards good stability.

Table 2 e The anodic peak currents of biological

analogues and their respective peak potentials.
Analyte Anodic Current (mA) Peak Potential (V)
Lysine 49 0.45
Glycine 52 0.67
Fig. 5 e Selectivity results of the as-fabricated ZnV2O4 Cysteine 78 0.5
MLNs. Comparison of inferring agents such as lysine, Glucose 22 0.25
glycine, cysteine, glucose, leucine, glutamic acid and Leucine 29 0.41
glutathione at 50 mM (each) with background analyte Glutamic acid 44 0.39
Glutathione 142 0.31
50 mM NaH2PO4. H2O and scan rate 100 mVs¡1.
1784 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5

Fig. 6 e Reproducibility and stability parameters of ZnV2O4 MLNs. (A) Reproducibility data of the ZnV2O4 MLNs sensor
towards 50 mM glutathione, and (B) Stability profile of ZnV2O4 MLNs sensor towards 50 mM glutathione; reaction conditions
were 50 mM NaH2PO4. H2O as the background media and scan rate 100 mVs¡1.

them. Sadia Z. Bajwa and Waheed S. Khan is responsible for

4. Conclusions the conceptualization, methodology, resource management,
and supervision of the study. All authors contributed equally
In conclusion, it is a great challenge to synthesize layered to the data analysis, discussion, and manuscript preparation.
structures as their synthesis is a complex and laborious task. Further, all authors reviewed the final version of the manu-
However, we adopted a very straight forward and cost- script and provided their consent.
effective, solution grown method. We were able to obtain
ZnV2O4 nanosheets, 15e20 nm thick, with a dense array of
multilayer network. This outstanding morphology ensures Declaration of Competing Interest
larger surface area and greater catalytic sites leading to
enhanced binding capabilities. The prepared ZnV2O4 MLNs The authors affirm no conflict of interest.
generate highly sensitive electrochemical signals, yielding a
limit of 9 nM. Remarkably, the sensor showed anodic and
cathodic signal separation with 40 mV that ensures a fast Acknowledgement
redox reaction towards glutathione. The sensor shows the
outstanding selectivity of glutathione in the presence of other This study is funded by research projects (Grant Number 6116,
structurally related biomolecules. This work can be extended 6117 and 7435). The authors acknowledge the Higher Educa-
to synthesize layered nanomaterials of assorted chemical tion Commission (HEC) of Pakistan for its -financial support.
compositions and morphology. Further, these structures can
be exploited for their binding capacity in various applications
as biosensors and catalysis, etc.
Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.jmrt.2022.01.110.
Novelty statement

In this manuscript, we have developed a facile yet versatile references

approach to fabricate dense layers of ZnV2O4 nanosheets
which are integrated with one another. The prepared interface
provides greater surface area for the catalysis of glutathione, [1] Lu J, Liu H. A critical review on the carrier dynamics in 2D
improving the planer surface of the sensor and the number of layered materials investigated using THz spectroscopy. Opt
binding sides leading to excellent sensitivity. To the best of Commun 2018;406:24e35.
[2] El-Borady OM, El-Sayed AF. Synthesis, morphological,
our knowledge, this is the first report where ZnV2O4 MLNs
spectral and thermal studies for folic acid conjugated ZnO
based interfaces are used to detect biomarkers. This work will
nanoparticles: potency for multi-functional bio-
open a new avenue to explore multilayer nanostructures for nanocomposite as antimicrobial, antioxidant and
the detection of important biomolecules. photocatalytic agent. J Mater Res Technol 2020;9:1905e17.
[3] Wang K, Ye Z, Liu C, Xi D, Zhou C, Shi Z, et al. Morphology-
controllable synthesis of cobalt telluride branched
Author contributions nanostructures on carbon fiber paper as electrocatalysts for
hydrogen evolution reaction. ACS Appl Mater Interfaces
2016;8(5):2910e6.
A. Taj, Rabisa Zia, Adnan Mujahid, Adeel Afzal performed
[4] Li L, Yang Y, Wang Y, Liang M, Huang Y. Electrochemical
material synthesis, characterization, sensor experiments. A. activity of layered double hydroxides supported nano Pt
Taj prepared the main document. A. Maqsood, Z. Tariq, clusters toward methanol oxidation reaction in alkaline
Faheem K. Butt synthesized the materials and characterized solutions. J Mater Res Technol 2020;9:5463e73.
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 2 ; 1 7 : 1 7 7 7 e1 7 8 5 1785

[5] Kim JU, Lee S, Kim T. Recent advances in unconventional [23] Lobo RFM, Pereira-da-Silva MA, Raposo M, Faria RM,
lithography for challenging 3D hierarchical structures and Oliveira Jr ON. In situ thickness measurements of ultra-thin
their applications. J Nanomater 2016;2016:56. multilayer polymer films by atomic force microscopy.
[6] Tian R, Sun J, Qi Y, Zhang B, Guo S, Zhao M. Influence of VO2 Nanotechnology 1999;10(4):389.
nanoparticle morphology on the colorimetric assay of H2O2 [24] Flexer V, Forzani ES, Calvo EJ, Luduen ~ a SJ, Pietrasanta LI.
and glucose. Nanomaterials 2017;7(11):347. Structure and thickness dependence of “molecular wiring” in
[7] Jian X, Wu B, Wei Y, Dou SX, Wang X, He W, et al. Facile nanostructured enzyme multilayers. Anal Chem
synthesis of Fe3O4/Gcs composites and their enhanced 2006;78(2):399e407.
microwave absorption properties. ACS Appl Mater Interfaces [25] Al-Attafi K, Nattestad A, Yamauchi Y, Dou SX, Kim JH.
2016;8(9):6101e9. Aggregated mesoporous nanoparticles for high surface area
[8] Kolli SK, Van der Ven A. Controlling the electrochemical light scattering layer TiO2 photoanodes in Dye-sensitized
properties of spinel intercalation compounds. ACS Appl Solar Cells. Sci Rep 2017;7(1):1e8.
Energy Mater 2018;1(12):6833e9. [26] Khan I, Saeed K, Khan I. Nanoparticles: properties,
[9] Kharel PL, Cuillier PM, Fernando K, Zamborini FP, applications and toxicities. Arab J Chem 2017;12:908.
Alphenaar BW. Effect of rare-earth metal oxide nanoparticles [27] Mao LJ, Liu CY, Li J. Template-free synthesis of VO x
on the conductivity of nanocrystalline titanium dioxide: an hierarchical hollow spheres. J Mater Chem
electrical and electrochemical approach. J Phys Chem C 2008;18(14):1640e3.
2018;122(27):15090e6. [28] Butt FK, Cao C, Wan Q, Li P, Idrees F, Tahir M, et al. Synthesis,
[10] Kong Y, Song Z, Wang S, Xia Z, Liu Q. Charge transfer, local evolution and hydrogen storage properties of ZnV2O4
structure, and the inductive effect in rare-earth-doped glomerulus nano/microspheres: a prospective material for
inorganic solids. Inorg Chem 2018;57(19):12376e83. energy storage. Int J Hydrogen Energy 2014;39(15):7842e51.
[11] Ding S, Mao J, Zeng X, Cheng X. Enhanced infrared emission [29] Butt FK, Tahir M, Cao C, Idrees F, Ahmed R, Khan WS, et al.
property of NiCr spinel coating doped with MnO2 and rare- Synthesis of novel ZnV2O4 hierarchical nanospheres and
earth oxides. Surf Coating Technol 2018;344:418e22. their applications as electrochemical supercapacitor and
[12] Bafaqeer A, Tahir M, Amin NAS. Synthesis of hierarchical hydrogen storage material. ACS Appl. Mater. lnterfaces
ZnV2O6 nanosheets with enhanced activity and stability for 2014;6(16):13635e41.
visible light driven CO2 reduction to solar fuels. Appl Surf Sci [30] Vinoth V, Wu JJ, Asiri AM, Anandan S. Sonochemical
2018;435:953e62. Synthesis of silver nanoparticles anchored reduced graphene
[13] Taj A, Zia R, Xu J, Younis S, Estrela P, Mahmood A, et al. oxide nanosheets for selective and sensitive detection of
Biogenic preparation of doughnut shaped manganese glutathione. Ultrason Sonochem 2017;39:363e73.
nanograins embellished on graphene for superior interfacial [31] Raoof JB, Ojani R, Karimi-Maleh H, Hajmohamadi MR,
binding of biomarkers. J Mater Res Technol 2020;9(5):9896e906. Biparva P. Multi-wall carbon nanotubes as a sensor and
[14] Gherab K, Al-Douri Y, Hashim U, Ameri M, Bouhemadou A, ferrocene dicarboxylic acid as a mediator for voltammetric
Batoo KM, et al. Fabrication and characterizations of Al determination of glutathione in hemolysed erythrocyte. Anal
nanoparticles doped ZnO nanostructures-based integrated Methods 2011;3(11):2637e43.
electrochemical biosensor. J Mater Res Technol [32] Beitollahi H, Gholami A, Ganjali MR. Preparation,
2020;9:857e67. Characterization and electrochemical application of AgeZnO
[15] Chinnakkannu Vijayakumar S, Venkatakrishnan K, Tan B. nanoplates for voltammetric determination of glutathione
SERS active nanobiosensor functionalized by self-assembled and tryptophan using modified carbon paste electrode. Mater
3D nickel nanonetworks for glutathione detection. ACS Appl Sci Eng C 2015;57:107e12.
Mater Interfaces 2017;9(6):5077e91. [33] Nesakumar N, Berchmans S, Alwarappan S. Chemically
[16] Kong X-J, Wu S, Chen T-T, Yu R-Q, Chu X. MnO2-induced modified carbon based electrodes for the detection of
synthesis of fluorescent polydopamine nanoparticles for reduced glutathione. Sensor Actuator B Chem
reduced glutathione sensing in human whole blood. 2018;264:448e66.
Nanoscale 2016;8(34):15604e10. [34] Pereira-Rodrigues N, Cofre  R, Zagal JH, Bedioui F.
[17] Cao N, Zeng P, Zhao F, Zeng B. Au@SiO2@RuDS Electrocatalytic activity of cobalt phthalocyanine CoPc
nanocomposite based plasmon-enhanced adsorbed on a graphite electrode for the oxidation of reduced
electrochemiluminescence sensor for the highly sensitive glutathione (GSH) and the reduction of its disulfide (GSSG) at
detection of glutathione. Talanta 2019;204:402e8. physiological pH. Bioelectrochemistry 2007;70(1):147e54.
[18] Nam H, Kwon JE, Choi M-W, Seo J, Shin S, Kim S, et al. Highly [35] Safavi A, Maleki N, Farjami E, Mahyari FA. Simultaneous
sensitive and selective fluorescent probe for ascorbic acid with electrochemical determination of glutathione and
a broad detection range through dual-quenching and bimodal glutathione disulfide at a nanoscale copper hydroxide
action of nitronyl-nitroxide. ACS Sens 2016;1(4):392e8. composite carbon ionic liquid electrode. Anal Chem
[19] Yang H, Liu A, Wei M, Liu Y, Lv B, Wei W, et al. Visual label- 2009;81(18):7538e43.
free telomerase activity monitor via enzymatic etching of [36] Keyvanfard M, Alizad K. A sensitive voltammetric sensor for
gold nanorods. Anal Chem 2017;89(22):12094e100. determination of glutathione based on multiwall carbon
[20] Heydari H, Gholivand MB, Abdolmaleki A. Cyclic nanotubes paste electrode incorporating pyrogallol. Red
voltammetry deposition of copper nanostructure on Orient J Chem 2014;30(2):593e9.
MWCNTs modified pencil graphite electrode: an ultra- [37] Abbas MN, Saeed AA, Ali MB, Errachid A, Zine N, Baraket A,
sensitive hydrazine sensor. Mater Sci Eng C 2016;66:16e24. et al. Biosensor for the oxidative stress biomarker
[21] Taj A, Shaheen A, Xu J, Estrela P, Mujahid A, Asim T, et al. In- glutathione based on SAM of cobalt phthalocyanine on a
situ synthesis of 3D ultra-small gold augmented graphene thioctic acid modified gold electrode. J Solid State
hybrid for highly sensitive electrochemical binding Electrochem 2019;23:1129e44.
capability. J Colloid Interface Sci 2019;553:289e97. [38] Karimi-Maleh H, Keyvanfard M, Alizad K, Khosravi V,
[22] Chen HA, Hsin CL, Huang YT, Tang ML, Dhuey S, Cabrini S, Asnaashariisfahani M. Electrocatalytic determination of
et al. Measurement of interlayer screening length of layered glutathione using multiwall carbon nanotubes paste
graphene by plasmonic nanostructure resonances. J Phys electrode as a sensor and isoprenaline as a mediator. Int J
Chem C 2013;117(43):22211e7. Electrochem Sci 2012;7(8):6816e30.