This document describes a study that assessed the antimicrobial properties of selenium nanoparticles (SeNPs) using cyclic voltammetry. SeNPs were synthesized using a microwave-assisted method with controllable size distributions. Cyclic voltammetry experiments showed that adding SeNPs to E. coli cells caused a continuous decrease in characteristic peak intensities over time, demonstrating a reduction in viable bacteria. This indicates that SeNPs interact with and disrupt the integrity of bacterial cell membranes, likely by forming reactive oxygen species, and can be used as potential antimicrobial agents.

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Vol. 19, 1–6, 2019
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Assessment of Antimicrobial Features of Selenium
Nanoparticles (SeNPs) Using Cyclic
Voltammetric Strategy
Madiha Saeed1 2 3
, M. Tayyab Ansari4
, Imdad Kaleem5
, Sadia Zafar Bajwa1
, Asma Rehman1
,
Khizra Bano1
, Bushra Tehseen1
, Nuzhat Jamil1
, Mehvish Zahoor1
, Ayesha Shaheen1
,
Ayesha Taj1
, M. Rizwan Younis6
, and Waheed S. Khan1 2 ∗
1
Nanobiotechnology Group, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road Faisalabad-38000, Pakistan
2
Biomaterials Research Group, Functional Materials and Nanodevices Division, Ningbo Institute of Materials Technology and
Engineering (NIMTE), Chinese Academy of Sciences (CAS), Ningbo City, Zhejiang 315201, P. R. China
3
University of Chinese Academy of Sciences, Shijingshan District, Beijing, 100049, P. R. China
4
Faculty of Pharmacy, University of Lahore (New Campus), Lahore, Pakistan
5
Department of Biosciences, COMSATS Institute of Information Technology (CIIT), Islamabad, Pakistan
6
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing, 210093, P. R. China
The emerging biomedical applications of selenium nanoparticles (SeNPs) require facile and efficient
strategy to assess its interactions with cell membrane. In this study, an efficient and reproducible
microwave assisted method was used to synthesize SeNPs with controllable size distributions.
The physical properties of the emergent structures, such as morphology, structure, and size were
studied. The antimicrobial applications of SeNPs were assessed by electrochemical analyses that
entailed the systematic acquisition of cyclic voltammetry data. Our results demonstrate a straight-
forward method to predict the integrity of bacterial cell membranes following the administration of
SeNP treatments.
Keywords: Selenium Nanoparticles, Modified Hydrothermal, Electrochemical Analysis,
Antimicrobial Features.
1. INTRODUCTION
Selenium (Se) is a fundamental trace element in the
human body. Se demonstrates both anti-oxidative and
pro-oxidative effects, which contribute significantly to
reinforcing competent cellular functioning, growth, and
nourishment, and that display critical features for advanc-
ing contemporary medicine.1
Se in its native form is
crucial for promoting good human health and for main-
taining several essential biological functions, especially
through the activation of seleno-enzymes such as glu-
tathione peroxidase.2
Evidence further supports the appli-
cability of Se supplements to impede the onset of, or
to treat, various medical conditions and diseases, includ-
ing arthritis, cardiovascular disease, cystic fibrosis, and
∗
Author to whom correspondence should be addressed.
muscular dystrophy.3
Many studies have revealed, more-
over, that increased levels of intracellular reactive oxygen
species (ROS) produced by the interactions of Se with bac-
terial cells could prompt desired cytotoxic effects and sup-
press the progression of disease-causing infections, such
as those caused by the buildup of bacterial biofilms along
surgically-delivered implants.4
With regards to many types
of mammalian cells, selenium nanoparticles (SeNPs) have
shown excellent biological activity, bioavailability, and
low toxicity.5
They can efficiently scavenge free radicals.6
SeNPs are additionally biodegradable and nontoxic, as
compared to metallic nanoparticles.7
SeNPs have been
reported as potential antimicrobial agents.4 6–8
Although
Se-enriched probiotics have demonstrated the capability
to inhibit the growth of the Gram-negative bacterium,
Escherichia coli (E. coli),9
the antimicrobial activities of
J. Nanosci. Nanotechnol. 2019, Vol. 19, No. xx 1533-4880/2019/19/001/006 doi:10.1166/jnn.2019.16627 1

2. Assessment of Antimicrobial Features of Selenium Nanoparticles (SeNPs) Using Cyclic Voltammetric Strategy Saeed et al.
SeNPs are comparatively less explored.10
Recent stud-
ies, however, provide evidence indicating the antimicrobial
properties of SeNPs.11–15
Cyclic voltammetry (CV) is widely adopted as an
electroanalytical technique in biochemical research.16
The
principles of CV can be adapted to the examination of
bacterial cell membrane destruction and subsequent death
as the result of SeNP exposure. There are various types of
bacteria that have the ability to utilize ferric iron [Fe(III)]
as their electron acceptor;17
the primary reason for this
is that about 80% of the membrane-bound cytochromes
found in bacteria are present in their outer membranes.18
Therefore, the objective of this research was to study
the interaction of SeNPs with bacterial cells using cyclic
voltammetry as an indicator of bacterial population varia-
tions. Our results demonstrate the promising use of the CV
technique to assess the bacterial cell response after their
exposure to SeNPs.
2. MATERIALS AND METHODS
Selenium nanoparticles (SeNPs) were synthesized by
using our previously reported method, with some
modifications.19
Briefly, the selenium dioxide (SeO2 pre-
cursor was converted into SeNPs through reaction at high
temperatures with 4-aminoantipyrine as a reducing agent
and polyvinylpyrrolidone (PVP) as a particle surface sta-
bilizer. Routinely, 4.0 ml of SeO2 (25 mM), 0.61 ml of
4-aminoantipyrine (100 mM), and 1.0 ml of PVP (5 mM)
were mixed in a reaction vial, which was placed in a
microwave reactor (CEM Discover Synthesizer) for 5 min-
utes under 100 W of applied microwave power. Different
molecular weights of PVP (Mw ∼ 29 kDa and 1,300 kDa)
and a series of reaction temperatures (50, 60, 70, 80, 100
and 150 C) were used in the synthesis of SeNPs hav-
ing different morphological features. After the termination
of the 5 minutes reaction period, a change in the color
of the solution, ranging from yellow to red depending on
temperature conditions during the synthesis, indicated the
formation of SeNPs. The product NPs were centrifuged
(Eppendorf 5424 Centrifuge) at 12,000 rpm for 40 minutes
and thoroughly washed with double distilled deionized
water and ethanol. Then, the pellet was dispersed in double
distilled deionized water for further characterization.
The synthesized products were characterized using vari-
ous analytical approaches, including X-ray powder diffrac-
tion (XRD, Philips X’Pert Pro MPD) with a Cu-K
radiation source ( = 0.15418 nm); field emission scan-
ning electron microscopy (FESEM), using the JEOL JSM-
7500F model, equipped with an auxiliary transmission
electron microscope (TEM); atomic force microscopy
(AFM, Shimadzu, SPS-9600); dynamic light scattering, for
the determination of particle surface charge and hydrody-
namic radius (Malvern ZEM-3600); and UV-visible spec-
troscopy, utilizing a NanoDrop 2000 spectrophotometer,
Figure 1. XRD pattern of selenium nanoparticles (SeNPs).
for the acquisition of data relating to characteristic par-
ticle absorption peaks. The antimicrobial features of the
SeNPs were assessed by electrochemical analysis, in par-
ticular, through the application of cyclic voltammetry in
connection with a P/G Stat (Autolab) user interface.
3. RESULTS AND DISCUSSION
Recall that SeNPs were synthesized using SeO2 as the
Se precursor and 4-aminoantipyrine as a reducing agent,
while polyvinylpyrrolidone (PVP) of different molecular
weights was used as a stabilizer. In order to investigate
the SeNP sample crystallinity, X-ray powder diffraction
(XRD) was performed. Figure 1 shows a typical XRD
pattern of trigonal Se nanoparticles. The diffraction peaks
correspond to the following Miller indices: (100), (101),
Figure 2. Transmission (A, B), scanning electron micrographs (C, D),
topographic images (E), and three-dimensional images (F) of SeNPs syn-
thesized at 80 C.
2 J. Nanosci. Nanotechnol. 19, 1–6, 2019

3. Saeed et al. Assessment of Antimicrobial Features of Selenium Nanoparticles (SeNPs) Using Cyclic Voltammetric Strategy
Figure 3. Digital photographic images of different sizes of SeNPs synthesized at different temperatures (A) and the corresponding UV-Vis absorption
spectrum (B).
(110), (102), (111), (200), (201), (112), (202), (210), (113),
and (301). All the sharp and strong diffraction peaks were
readily correlated to the trigonal structure of Se nanopar-
ticles, which consistently yield an average lattice constant
of = 4.35 Å following X-ray analysis; this is in good
agreement with the data documented in the JCPDS stan-
dard card (No. 06–0362) for SeNPs.
The transmission and scanning electron micrographs
showed the presence of spherical SeNPs having an average
Figure 4. Field emission scanning electron micrographs (FESEM) of as-synthesized SeNPs (A–C) at 50 C; (D–F) at 60 C; and (G–I) at 70 C.
diameter of approximately 360–380 nm following their
formation at 80 C (Figs. 2(A–D)). The SeNPs were fur-
ther analyzed using atomic force microscopy (AFM) in
direct contact mode (Fig. 2(E)), and the procured three-
dimensional images of the SeNP surfaces showed a maxi-
mum height of approximately 72 nm (Fig. 2(F)).
By physical and visual interpretations, it was concluded
that different reaction temperatures produced SeNP sus-
pensions with distinctive bulk appearances, especially in
J. Nanosci. Nanotechnol. 19, 1–6, 2019 3

4. Assessment of Antimicrobial Features of Selenium Nanoparticles (SeNPs) Using Cyclic Voltammetric Strategy Saeed et al.
terms of observed color, as shown in Figure 3(A). The
UV-visible spectrum of the SeNPs was measured to assess
absorption in the visible range. The obtained spectrum
showed the characteristic absorption peak of SeNPs at
205 nm (Fig. 3(B)).
The color change from light yellow to deep red at differ-
ent reaction temperatures (50–100 C) indicated the forma-
tion of different sizes of SeNPs. Notably, it was observed
that light yellow particle suspensions appeared at 50 C,
which correlated with smaller SeNPs, and that color inten-
sities were incrementally amplified as reaction tempera-
tures were raised up to 100 C and as particle diameters
increased. Further accretion in the reaction temperature up
to 150 C results in particle aggregation. Therefore, it is
indicated that the increase in temperature results in the
formation of larger particles. Field emission scanning elec-
tron micrographs of as-synthesized SeNPs at 50, 60, and
70 C are shown in Figure 4.
Zeta-potential data, depicted in Figure 5(A), shows the
presence of a negative surface charge on the synthesized
SeNPs, and additional DLS analysis indicates average par-
ticle hydrodynamic diameters of 311 nm and 663 nm, cor-
responding to reaction temperatures of 50 C and 80 C,
respectively (Figs. 5(B and C)).
The effects of SeNPs on E. coli cells were studied
by conducting cyclic voltammetry experiments. Figure 6
shows the output cyclic voltammograms, which were
obtained first by completing scans of the background
media and then by adding SeNPs to the reaction medium.
Figure 5. Zeta potential (A) and number distribution data of SeNPs synthesized at 50 and 80 C, respectively (B, C).
The addition of SeNPs caused no significant change in the
CV output, relative to the background reading, and this
result validates the noninterference of SeNPs with the elec-
trodes. Afterwards, E. coli cells were introduced into the
standard test vessel, and scans were recorded immediately
following bacterial inoculation at time t = 0, as well as
after two different incubation periods, of 20 minutes and
24 hours, to discriminate the interaction of SeNPs with
E. coli.
At increasing incubation times, a continuous decrease in
characteristic peak intensities was observed, demonstrating
the progressive reduction in the number of viable bacteria.
The graph shown in Figure 7 displays a distinct decrease
in current intensity with the passage of time.
Generally, bacterial cell membranes consist of redox
active proteins and electrically conductive pilli. However,
these proteins are electrochemically inactive due to the
presence of non-conducting material such as peptidogly-
can and lipids. A cyclic voltammeter can be utilized to
characterize and detect the activities of redox proteins like
cytochrome. A mediator such as Fe(CN6
3−
can be used to
facilitate the electron transfer between electrochemically
inactive microorganisms and an electrode.20
In such cases,
electrochemical oxidation of ferricyanide provided elec-
trochemical data relating to the quantity of bound cells.
Thus, the degree of the reduction of water insoluble ferri-
cyanide by viable E. coli cells is an indicator of the extent
of the redox interactions encountered by the microorgan-
isms at any given time point and is particularly sensitive
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5. Saeed et al. Assessment of Antimicrobial Features of Selenium Nanoparticles (SeNPs) Using Cyclic Voltammetric Strategy
Figure 6. Cyclic voltammograms showing the response of E. coli inter-
actions with SeNPs at different incubation times (t = 0, 20 min, and
24 h).
to their growth conditions. Figure 6 illustrates that SeNPs
are not involved in any systemic electrochemical reactions,
and for this reason, it is speculated that their addition does
not bring any change in the CV scan output, as is also
the case with the background medium. When E. coli is
introduced into the electrochemical cell along with SeNPs,
following extended incubation periods, the redox peaks
of ferricyanide undergo significant changes, corroborating
the interaction of SeNPs with bacterial cells. Cytochrome
localized on the outer bacterial membranes facilitates indi-
rect electron transfer to the CV electrodes. Thus, after
the influx of SeNPs, a decrease in the current response
indicates the presence of less intact and viable cells. NPs
can adsorb onto, or attach to anionic bacterial cell mem-
branes by electrostatic interactions. After the administra-
tion of NPs, reactive oxygen species (ROS) are formed.8
As a result, NPs are capable of disrupting the integrity
of bacterial cell membranes. Nanoparticles accumulate on
and endocytose across the membranes of E. coli cells and
effectively exhibit antibacterial effects due to the impair-
ment of external structures or internal mechanisms.21
It
is reported that SeNPs are able to disturb the integrity
of the bacterial cell membrane12
due to the formation of
ROS. As a result, bacterial cells undergo cell lysis.22 23
It
is plausible that SeNPs are first adsorbed onto the outer
cell membrane of the bacteria before penetrating the mem-
brane and causing cellular lysis. As the result of this, less
Figure 7. The magnitude of anodic currents obtained from CV data is
plotted against incubation time.
cytochromes of intact bacteria are available for the reduc-
tion of ferricyanide, and a decrease in the anodic and
cathodic current is observed. To supplement this supposi-
tion, anodic peak currents have been calculated and plot-
ted against incubation times (Fig. 7). From the data, it is
concluded that current intensities are dependent upon the
length of the incubation term during which the microor-
ganisms are in contact with SeNPs. When the incubation
time is increased, the oxidation and reduction of iron is
gradually decreased, and this validates the disruption of
the cellular membrane integrity following SeNP exposure.
4. CONCLUSIONS
In summary, we have synthesized SeNPs by an efficient
and facile method. We adapted the cyclic voltammetry
method to assess the integrity of bacterial cell mem-
branes. Our results successfully demonstrated that bacte-
rial cells undergo cell lysis due to exposure to SeNPs. In
the future, this method can be used to study the interac-
tions of nanoparticles with different cell membranes and
to assess the antimicrobial activities of various kinds of
nanoparticles.
Acknowledgments: This work was supported by the
Higher Education Commission (HEC) of Pakistan through
Research Grant No. 6115. Dr. Waheed S. Khan also
acknowledges the support of Chinese Academy of Sci-
ences under CAS-PIFI Fellowship at Ningbo Institute of
Materials Technology and Engineering (NIMTE), Ningbo
City, Zhejiang, P. R. China. Ms. Madiha Saeed thanks
the Chinese Academy of Sciences (CAS) and The World
Academy of Sciences (TWAS) for awarding her a CAS-
TWAS President’s Ph.D. fellowship (2014A8017407006).
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Received: 4 April 2018. Accepted: 23 July 2018.
6 J. Nanosci. Nanotechnol. 19, 1–6, 2019