Elucidating the chemical and biochemical applications of Citrus sinensis-mediated silver nanocrystal

Article abstract of DOI:10.1080/07391102.2018.1559763

Synthesis of nanoparticles using biodegradable source is safer and echo-friendly. Here, we describe the synthesis of polycrystalline silver nanocrystals using Citrus sinensis acting as both reducing and capping agents. After exposing the silver ions to orange extract, rapid reduction of silver ions led to the formation of stable silver nanocrystals due to the reducing and stabilizing properties of orange fruit juice. The synthesized silver nanocrystals were characterized using various analytical techniques like UV–vis spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscopy (TEM). The biochemical activity of the synthesized nanocrystals was studied in the light of affinity to bovine serum albumin using several biophysical methods like absorbance, fluorescence and circular dichroism spectroscopy. Cytotoxic activity of these nanocrystals was also studied against Hep-2 cell line using fluorescence microscopy. It was also found that the synthesized nanocrystals can sense mercuric ion down to 50 μM in the presence of a number of cations. Furthermore, we established that the silver nanoparticles can effectively catalyse the reduction of methylene blue by ascorbic acid. The present study will enrich our knowledge on the chemical and biochemical activities of green-synthesized silver nanocrystals.

Full text of DOI:10.1080/07391102.2018.1559763

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Elucidating the chemical and biochemical
applications of Citrus sinensis mediated silver
nanocrystal
Nurunnesa Siddiqui, Abdulla Al Masum, Md. Raihan Uddin, Sukhendu
Mandal, Mohana Sengupta, Md. Maidul Islam & Subrata Mukhopadhyay
To cite this article: Nurunnesa Siddiqui, Abdulla Al Masum, Md. Raihan Uddin, Sukhendu Mandal,
Mohana Sengupta, Md. Maidul Islam & Subrata Mukhopadhyay (2018): Elucidating the chemical
and biochemical applications of Citrusꢀsinensis mediated silver nanocrystal, Journal of Biomolecular
Structure and Dynamics, DOI: 10.1080/07391102.2018.1559763
To link to this article: https://doi.org/10.1080/07391102.2018.1559763
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Elucidating the chemical and biochemical applications of Citrus
sinensis mediated silver nanocrystal
a
b
c
c
Nurunnesa Siddiqui , Abdulla Al Masum , Md. Raihan Uddin , Sukhendu Mandal ,
a
d
a
Mohana Sengupta , Md. Maidul Islam* , Subrata Mukhopadhyay
a
Department of Chemistry, Jadavpur University, Kolkata 700032, West Bengal, India
b
Nanobiotechnology Laboratory, Department of Life Science and Biotechnology, Jadavpur
University, Kolkata 700032, West Bengal, India
c
Department of Microbiology, University of Calcutta, 35, B. C. Road, Ballygunge, Kolkata
7
00019, West Bengal, India
d
Department of Chemistry, Aliah University, Action Area IIA/27, New Town, Kolkata 700156,
West Bengal, India
1

Abstract
Synthesis of nanoparticles using biodegradable source is safer and echo-friendly. Here we
describe the synthesis of polycrystalline silver nanocrystals using Citrus sinensis acting both
as reducing and capping agent. After exposing the silver ions to orange extract, rapid
reduction of silver ions led to the formation of stable silver nanocrystals due to the reducing
and stabilizing properties of orange fruit juice. The synthesized silver nanocrystals were
characterized using various analytical techniques like UV–Vis spectroscopy, XRD, Dynamic
Light Scattering (DLS), SEM and TEM. The biochemical activity of the synthesized
nanocrystals was studied in the light of affinity to Bovine Serum Albumin using several
biophysical methods like absorbance, fluorescence and circular dichroism spectroscopy.
Cytotoxic activity of these nanocrystals was also studied against Hep–2 cell line using
fluorescence microscopy. It was also found that the synthesized nanocrystals can sense
mercuric ion down to 50 µM in presence of a number of cations. Furthermore, we established
that the silver nanoparticles can effectively catalyze the reduction of methylene blue by
ascorbic acid. The present study will enrich our knowledge on the chemical and biochemical
activity of green–synthesized silver nanocrystals.
Keywords: Green synthesis, Silver nanocrystals, Orange extract (Citrus sinensis), Protein
binding, Antibacterial activity, Cytotoxicity study, Mercury sensing, Catalytic activity.
2

Introduction
Nanoparticles speak for themselves due to their specific physicochemical properties that
include optical, magnetic and catalytic property, among others (Liu, 2005; Cao, 2005; Quinn,
2
005; Sun, 2006; Li, 2006; Noubactep, 2005; Feng, 2007; Wang, 1997). Nanoparticles may
be amorphous or crystalline. The crystalline shape of nanoparticles is called nanocrystals.
They are much effective in catalysis, biosensing, imaging, drug delivery, nanodevice
fabrication and in medicine (Nair, 2007). There are numerous methods for the synthesis of
silver nanocrystals that include physical, chemical and green synthetic methods (Lalitha,
2
013). Among these protocols, physical and chemical syntheses have toxic effects compared
to green synthetic methods (Jain, 2009). The requirement for environmental non–toxic echo
friendly protocols for nanocrystals synthesis has a ever-demanding interest as such
approaches are free from the use of toxic chemicals. Thus, there is a rising quest for green
synthesis of nanomaterials (Singhal, 2011; Sathya, 2012). The green synthetic method (like
that we report here) may be the best for the synthesis of silver nanocrystals as it is an eco–
friendly approach (Sathya, 2012). Nanocrystalline silver particles have been establishing
fabulous applications in the fields of high sensitivity biomolecular detection, diagnostics,
antimicrobials, therapeutics, catalysis and micro–electronics (Anjum, 2016).
Silver is well known for possessing an inhibitory effect headed for many bacterial strains and
microorganisms, usually present in medical and industrial processes (Jiang, 2004). In
medicines, silver and silver nanoparticles have a plentiful function as well as skin ointments
and creams containing silver are used to prevent infection of burns and open wounds (Durán,
2
005), in medical devices and in implants prepared with silver–impregnated polymers
(Becker, 1999). A lot of plant extracts had been used to synthesize silver and gold
nanoparticles (Kumar, 2009; Mittal, 2013; Mie, 2013) but the literature are not so rich on the
use of fruit extracts for the synthesis of nanopartlces (Jain, 2009). Many fruit extracts contain
3

polyhydroxyl groups (Vámos‐Vigyázó, 1981) and they are liable for the reduction of silver
ions, whereas water soluble heterocyclic components in it can stabilize the synthesized silver
nanocrystals (Roco, 1999;Chan, 2002; Thess, 1996; Zhan, 2003; Linsebigler, 1995; Zhang,
1
999; Service, 1998; Rosi, 2006; chukin, 2003). The major benefit of biological methods is
the availability of amino acids, proteins, or secondary metabolites present in the synthesis
process. Extra step required for the avoidance of particle aggregation, using plant extracts as
reducing agents, it can be possible to control the shape, size, and monodispersity of the
nanocrystals (Gurunathan, 2014; Gurunathan, 2009).
Nanoparticles have high affinty towards proteines which may cause drastic effect in living
cells although this property can be used to terget specific proteine in the field of
nanomedicine (Beg, 2018; Beg, 2018). Cancer cells grow in uncontrolled manner and spery
cancer in body due to their aggressive nature. The cytotoxic properties of nanoparticles
against cancer cells have the potential to design nano mediated anticancer drug (Braydich-
Stolle, 2005) in future. In present days several heavy metals like mercury is polluting our
environment; and sensing of heavy metals in environment is an urgent need. Nanoparticels
can sense several heavy metals (Kim Y, 2001; Lin, C. Y., 2010).
Mthelyne blue dye is extensively used in textile industries and pollutes nearby waterbodies.
Several reagents like alikaline glucose solution, ascrobic acid, citric acid, etc. can be used to
reduce such dyes. Nanoparticles can enhance the speed of such reaction acting as catalyst.
(Edison, T. J. I., 2012).
In the present work we describe a convenient, and green process to synthesize silver
nanocrystals using Citrus sinensis as both reducing and capping agent. We also explored the
protein binding, cytotoxic, Hg-sensing and catalytic properties of the nanocrystals to enrich
our knowledge in the field of nanoscience.
Materials and methods
4

Materials
To prepare orange extract (Citrus sinensis), fresh orange samples were collected from local
market. Silver nitrate and ascorbic acid (both of reagent grade) were procured from Merck
India Limited. Bovine Serum albumin (BSA) and Methelyne Blue were purchased from
Sigma–Aldrich and used as received.
Cell culture
Hep–2 Cell (ATCC: CCL–23) lines were prepared from continuous culture in Dulbecco’s
Modified Eagle’s Medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented with
1
0% fetal bovine serum (Invitrogen), penicillin (100 μg/mL), and streptomycin (100 μg/mL).
The Hep–2 were obtained from the American Type Culture Collection (Rockville, MD) and
maintained in DMEM containing 10% (v/v) fetal bovine serum and antibiotics in a CO2
2
incubator. Cells were initially propagated in 75 cm polystyrene, filter–capped tissue culture
flask in an atmosphere of 5% CO and 95% air at 37°C in CO incubator. When the cells
2
2
5
reached the logarithmic phase, the cell density was adjusted to 1.0 × 10 per/well in culture
4
media. The cells were then used to inoculate in a glass bottom dish, with 1.0 mL (1.0 × 10
cells) of cell suspension in each dish. After cell adhesion, culture medium was removed. The
cell layer was rinsed twice with phosphate buffered saline (PBS), and then treated according
to the experimental need.
Orange fruit extract preparation
Orange extract (Citrus sinensis) was obtained by smashing the orange inside a grinder with
1
00 mL of deionized water and the extract was filtered through a cheese cloth. This filtrate
was treated with equal volumes of deionized boiled water and the resultant precipitate was
centrifuged at 10000 rpm for 5 min. This was air–dried to a powder and used for further
experiments.
Synthesis of silver nanocrystals
5

For all experiments, the source of the silver was silver nitrate (AgNO ) in deionized water.
3
Typical reaction mixtures contained 50 mL of orange extract in 50 mL of silver nitrate
solution (0.05 M) unless otherwise stated. The mixture was stirred using magnetic stirrer at
8
0°C for nearly 7 hours until the colour of the solution change from pale yellow to deep
brown indicating formation of Ag nano. The reaction mixtures were monitored at different
time intervals and then the synthesized nanocrystals were characterized. The orange extract
content was varied while keeping the silver nitrate concentration at a level of 0.05 M. To
study the effect of temperature on nanocrystal synthesis, reaction mixtures containing orange
extract, and 0.05 M AgNO at pH 7.0 were incubated at 40, 60, 80 or 100 °C.
3
Characterization of silver nanocrystals
The formation of silver nanocrystals was investigated by scanning the original mixture under
Shimadzu Pharmaspec UV–1700 spectrophotometer connected with a PC (Shimadzu
Corporation, Tokyo, Japan) in the wavelength range between 300 and 700 nm after 400 mins
of incubation period. To study the mechanism of nanocrystal synthesis, the chemical
composition of the both orange extract (dry condition) and synthesized silver nanocrystal
(dry) was characterized by using FTIR spectrometer (Perkin Elmer Spectrum Version 10.4.1
-1
spectrometer) in the range 4000–400 cm . X–ray diffractometer (Ultima–III, Rigaku, Japan,
λ = 1.54 Å) was used to identify proper phase and crystallinity of the synthesized silver
nanocrystals. The surface morphology of nanocrystal was investigated using SEM (INCA
Pental FETX3). Transmission electron microscopy (TEM, 200 kV, JEOL–2010) was used to
know the shape, size and internal morphology of the synthesized nanocrystals. A couple of
drops of silver nanocrystal suspension (concentration 50 μg/mL) was placed on carbon coated
copper grid and dried inside a vacuum dryer. The copper grid was then scanned by TEM to
examine the nanocrystals. To prepare the sample for DLS analysis, the dried powder of silver
nanocrystals was suspended in deionized water maintaining a certain concentration (around
6

5
0 μg/mL). The hydrodynamic particle diameter and distribution of biosynthesized silver
nanocrystals was determined by dynamic light scattering using a Malvern Zetasizer
NanoZS90, Malvern Instruments Limited, Worcestsershire, U.K.).
(
Determination of molar extinction coefficient
The molar extinction coefficient of silver nanocrystals in water was determined using the
equation (Navarro, 2013).
ꢀ ꢁꢂ
where A = 2.3 × 10 M cm , γ = 3.48, d ≤ 38 nm and A = 4.8 × 10 M cm , γ = 0.77, d ≤
8 nm as in our case we found d is near about 164 nm the value if molar extinction
1
5
−1
−1
8
−1
−1
3
1
0
−1
−1
coefficient in our case is 2.13 × 10 M cm (Moores, 2006).
Protein binding study using spectroscopy
Absorbance spectral measurements were performed on a Shimadzu Pharmaspec UV–1700
spectrophotometer connected with a PC (Shimadzu Corporation, Tokyo, Japan) in 1.00 cm
path length strain–free matched quartz cells. Temperature of the cell holder was controlled by
a Lab companion water bath. Steady state fluorescence spectra were measured on a Shimadzu
RF5301PC spectrofluorimeter (Shimadzu Corporation, Tokyo, Japan) using excitation and
emission band pass of 3 nm. For measurement of intrinsic fluorescence of BSA in presence of
nanocrystals, the protein sample was excited at 295 nm, the excitation maximum of
tryptophan, and emission spectra was scanned from 300 to 400 nm.
Cell imaging study
4
For confocal imaging studies, 1 × 10 Hep–2 cells in 1000 μL of medium, were seeded on
sterile 35 mm glass bottom culture dish (ibidi GmbH, Germany), and incubated at 37 °C in a
CO incubator for 10 hours. Then cells were washed with 500 μL DMEM followed by
2
incubation with 0.05 M AgNPs dissolved in 500 μL DMEM at 37°C for 30 minutes in a CO2
incubator and observed under an Olympus IX81 microscope equipped with a FV1000
7

confocal system using 1003 oil immersion Plan Apo (N.A. 1.45) objectives. Images obtained
through section scanning were analyzed by Olympus Fluoview (version 3.1a; Tokyo, Japan)
with excitation at 350 nm monochromatic laser beam, and emission spectra were integrated
over the range 470 nm (single channel).
Cytotoxicity assay
The cytotoxic effects of the compound silver nanocrystals was determined by MTT [3–(4,5–
dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide] assay following the manufacturer’s
instruction (MTT 2003, Sigma–Aldrich, MO). Hep–2 cells were cultured into 96–well plates
4
(
10 cells per well) for 24 h. After overnight incubation, the medium was removed, and
various concentrations of silver nanocrystals (0–150 μM) made in DMEM were added to the
cells and incubated for 24 hrs. Cells without any treatment and cell–free medium were also
included in the study. Following incubation, the growth medium was removed, and fresh
DMEM containing MTT solution was added. The plate was incubated for 3−4 hrs at 37 °C.
Subsequently, the supernatant was removed, the insoluble colored formazan product was
solubilized in DMSO, and its absorbance was measured in a micro plate reader (Perkin–
Elmer) at 570 nm. The assay was performed in triplicate for each concentraion of the silver
nanocrystal compound. The absorbance value of wells containing only DMEM medium was
subtracted from all readings to get rid of the background influence. The cell viability was
calculated by the following formula: (mean absorbance in treated wells / mean absorbance in
control wells) × 100 (Sathishkumar, 2018).
Catalytic activity on methylene blue degradation
1
g Methelyne Blue was dissolved in 100 mL water to prepare the solution and distributed in
three beakers. 3 nM nanocrystal, 0.01 M ascorbic acid and a mixture of 3 nM nanocrystal and
.01 M ascorbic acid added in this beakers respectively. Then all the beakers were kept in
0
normal light and normal temperature to observe the reduction of MB. The spectral and kinetic
8

measurement was also executed in details (Soto-Robles, 2018).
Results and discussions
Formation of silver nanocrystals studied by UV–Vis spectroscopy
Orange extract was added to the silver nitrate solution and stirred for 400 mins. During this
period the colour of the solution changed distinctively from orange to dark brown (shown in
+
Fig. 1) indicating formation of silver nanocrystals. Orange extract rapidly reduces Ag ions to
produce silver nanocrystals and the process was monitored by UV–Vis spectroscopy (Fig. 2).
An absorbance peak at 277 nm with a hump nearly at 323 nm (the small arrow in Fig. 2) was
observed for orange extract. Growth of a peak at 416 nm was observed due to the formation
of silver nanocrystals. The absorbance increased with increasing time. No shift of this peak
position over time clearly indicated that there was no variation in particle size with time.
Dynamic light scattering analysis
The size distribution profile determined by dynamic light scattering (DLS) study indicates
that the size (diameter) of these silver nanocrystals was near about 200 nm which is near
about the length of the nanocrystal (Table 1). The high intensity of the distribution indicates
that the most of the synthesized particles are in the same range of particle size (Fig. 3).
Fourier-transform infrared spectroscopy:
The functional groups present in orange extract (also, capping agent) were identified using
-1
FTIR spectroscopy. Orange extract shows broad spectrum at 3308 cm which corresponds to
-1
alcoholic, phenolic or carboxylate -O-H stretching. Shifting of that peak to 3350 cm in
nanocrystal clearly indicated that alcoholic or phenolic group take part in synthesis of
nanocrystal. Orange extract shows peaks at 2929, 2115, 1715, 1616, 1522, 1404, 1341, 1196,
-1
1
022, 815 and 773 cm which may be due to the presence of –C-H, C≡C, C=O, C=C, N-O
of nitro group, C-C, alcoholic C-O group respectively (see figure 4). The changes in peak
9

-1
position, from 1341 to 1324 and from 1022 to 1016 (all in cm ) after silver nano formation
indicated that nitro group also take part in synthesis and capping of nanocrystal (Banala,
2
015).
XRD analysis
The X–ray diffraction pattern of the silver nanocrystals were characterized by powder
diffraction pattern and Miller Indices (h, k, l) to each peak was assigned as usual (El-Shamy,
2
018; Bose, 2013). Absence of any spurious diffraction confirmed that no crystallographic
impurities were present in the sample. The silver nanocrystals showed six peaks of silver at
2
θ = 27.8°, 33.4°, 38°, 44.3°, 64.8°, 77.5° that can be assigned to the (100, 110, 111, 200,
2
20, 311) faces of silver (Fig. 5), which resembles quiet well with the values obtained for the
FCC cubic structure of silver nanocrystals (JCPDS, File No. 43–0649) (Bose, 2013).
Small size and high degree of crystalline nature of the silver nanocrystals were identified by
broad spectrum and intensity of peaks respectively (Wani, 2011). The average size of silver
nanocrystals was determined by the Debye–Scherrer formula (John, 2009).
ꢃꢄꢅꢆ
D =
(2)
ꢇꢈꢉꢊꢋ
where λ is the wavelength, 0.154 nm, β is the half peak width in radian, θ is the diffraction
angle. The size of nanocrystals thus found was in the range of 11–15 nm, the average of
which was taken as 13 nm (Table 2).
1
0

Determination of Crystalinity
It was proved that sharper is the peak the larger is the size of the crystal. In our case
broadening of the XRD peaks were observed and the average particle size determined by
XRD was 13 nm. From this data and the data from DLS (size 211.2 nm) the crystalinity was
determined using Scherrer equation (Zak, 2011). as follows:
ꢌꢍꢎꢏꢌꢐꢑꢒꢎ
I =
cry
(3)
ꢌꢈꢓꢔꢊꢎꢏꢕꢖꢌꢒ
where I cry is crystallinity index, DP (DLS) is size of nanocrystal obtained from DLS (211.2
nm) and Dcrys is the size of crystal obtained from XRD (13 nm). In our study we observed
that crystallinity index Icry = 16.24 which clearly indicate that the nanocrystals are
polycrystalline in nature.
Scanning Electron Microscopy
Scanning Electron Microscopy was used for revealing the surface morphology of particles.
Here, the bead for the SEM analysis was prepared by placing a drop of the silver nanocrystal
suspension on the carbon tape attached to the head of cylindrical bead and it was dried in a
vacuum dryer for a couple of hours. The particles on the top of the bead were scanned by
Scanning Electron Microscope and we observed pillar shape, crystalline structure of silver
nanocrystals (Fig. 6).
Transmission electron microscopy
Transmission electron microscopy was used to investigate the microstructure and crystallinity
of silver nanocrystals. It is clear from the high resolution TEM image (Fig. 7) that our
prepared silver nanocrystals possess pillar shaped (square base) structure with an average
diameter nearly 164 nm. Particle size as obtained from TEM closely matches with that
obtained from DLS and SEM.
1
1

Biochemical application of silver nanocrystal
Bioactivities of the silver nanocrystals such as affinity towards protein molecules (Bovine
Serum Albumin, BSA), antimicrobial activity and Cytotoxicity were studied by several
methods, as described below.
Spectrophotometric study
Pure Bovine Serum Albumin (BSA) shows a peak 278 nm. The decreasing
absorbance of BSA with increasing concentration of silver nanocrystals with no shift of peak
position clearly indicated that BSA was adsorbed on the surface of silver nanocrystals
without changing the chemical composition of it (Cedervall, 2007; Xie, 2003; Das, 2018).
The binding constant was determined using Benesi–Hildebrand equation using the following
formula:
ꢗ
ꢗ
ꢗ
ꢀ
ꢞ
ꢎ
(4)
ꢀ
ꢘ
ꢏꢙꢚꢄꢀꢘꢃꢒꢛꢜꢍꢝꢎ
ꢀꢘꢃ
where ꢀA is absorbance/fluorescence intensity change, K is the binding constant, and ꢀA is
a
0
maximum absorbance/fluorescence intensity change. The binding constant was determined
by plotting 1/ꢀA vs. 1/[NP] and then dividing the intercept (c) by the slope (m) as is shown in
–1
Fig. 8 (inset). The resulting binding constant K (= c/m) was found to be 4685.44 uM (4.68 ×
9
–1
1
0 M ).
Spectrofluorometric study
The effect of silver nanocrystals on the fluorescence intensity of Bovine Serum Albumin
(BSA) is illustrated in Fig. 9 showing the steady state fluorescence emission spectra of
Bovine Serum Albumin (BSA) in the control (where silver nanocrystals are absent) and in the
presence of increasing concentration of silver nanocrystals. Upon excitation at 295 nm, the
changes in the fluorescence intensity of Bovine Serum Albumin (BSA) derived solely from
1
2

the tryptophan residues and this has previously been used extensively to study the interaction
of Bovine Serum Albumin (BSA) with small molecules (Das, 2018).
In the Bovine Serum Albumin (BSA), there are two tryptophan residues: tryptophan–134,
located near the protein surface buried in a hydrophilic pocket of domain I and tryptophan–
2
12, buried at an internal part of domain II. At 280 nm excitation, both tryptophan and
tyrosine are excited, but because of efficient resonance energy transfer from tyrosine to
tryptophan, most of the fluorescence comes from tryptophan–214. However, on excitation at
2
95 nm, the entire fluorescence intensity comes from tryptophan–214.
In presence of silver nanocrystals, the intrinsic fluorescence spectra of Bovine Serum
Albumin (BSA) were remarkably quenched, which might arise from the change in protein
conformation probably due to protein adsorption onto the surface of silver nanocrystals,
reflecting the relative changes in the proximity between tryptophan (active fluorescence
emitter) in the protein and the silver nanocrystals (the quenching agent). The binding constant
was determined using Benesi Hildebrand equation in the usual way. The resulting binding
9
–1
constant was found to be, k = c/m = 4.35 × 10 M .
1
3

Conformational aspects of binding
A large negative ellipticity near 208 and 222 nm (CD spectra, Fig. 10) due to π→π*
and n→π* indicate presence of the alpha helix in Bovine Serum Albumin (BSA). We
calculated the amount of alpha helix in BSA using the method of Kellys et al (Kelly, 2005).
From our experiment we found that initially 46% present of alpha helix in BSA. With
concentration of nanocrystals, the elliptitcity near 208 and 220 nm was decreased, which
clearly indicated distortion of the alpha helix due to interaction.
Cell imaging
The cells treated with silver nanocrystals gave bright blue fluorescent light (Singh,
2
012; Anand, 2015). While the control cell showed no such fluorescence intensity. Therefore,
these results clearly indicate that silver nanocrystals successfully penetrate inside the cells
Fig. 11).
(
Cytotoxicity assay
Silver nanocrystals showed less cytotoxic activity against Hep–2 cells when treated with
different concentrations in the range 0 – 100 μM for 24 hrs and cell survival was determined
by MTT assay (Singh, 2012; Anand, 2015). As seen in Fig. 12, cell survivability decreased
with increasing concentrating of silver nanocrystals. The Cell viability was determined using
Cell viability equation using the following formula:
ꢨꢠꢤꢩꢎꢤꢥꢪꢫꢬꢥꢤꢩꢭꢠꢎꢣꢩꢎꢦꢬꢠꢤꢦꢠꢮꢎꢯꢠꢡꢡꢪ
ꢨꢠꢤꢩꢎꢤꢥꢪꢫꢬꢥꢤꢩꢭꢠꢎꢣꢩꢎꢭꢫꢩꢦꢬꢫꢡꢎꢯꢠꢡꢡꢪꢒ
(
5)
ꢟꢠꢡꢡꢎꢢꢣꢤꢥꢣꢡꢣꢦꢧ ꢀ
ꢰꢱꢲꢲ
Catalytic activity on methylene blue reduction
Methelyne Blue (MB) is a blue thionin dye used for several purposes in cloth industry
1
4

(Rafatullah, 2010). Under acidic conditions, it is easily reduced to the colorless hydrogenated
+
molecule leucomethylene blue (LB ) by ascorbic acid (H A) as shown in Fig. 13 (Mowry,
2
1
999). The reduction procedure was studied in the presence of silver nanocrystals alone,
ascorbic acid alone (0.1 M) and a mixture of silver nanocrystals and ascorbic acid.
It was observed that in presence nanocrystals there was no change in colour of MB and
spectral pattern or intensity even after 1 hour of incubation. In the presence of ascorbic acid,
MB showed slow, stepwise decolorisation. With a mixture of ascorbic acid, silver
nanocrystals and MB, fast depolarization was observed. From UV visible spectroscopy it was
also observed that absorbance of MB decreased (Fig. 14) in both the cases, without any
shifting of max
.
To study the kinetics of reduction, the change of absorbance at 650 nm was recorded with
time. From the slope of log(Abs) vs. time plot we found that the rate constants presence of 0,
–
4
–4
–4
–4
0
.065, 0.65 and 6.5 nM of nanocrystal were 2.5 × 10 , 3.5 × 10 , 4.9 × 10 and 11.6 × 10
–1
in s respectively (Fig. 15).
Mercury Sensing
To study the cation sensing properties of the nanocrystals, we used the method stated by
Farhadi et al (Farhadi, 2012). Several salt solutions like HgCl , CoNO , NaCl, ZnCl CuSO
2
3
2,
4,
NiCl and MgCl was individually treated with nanocrystal solution (Fig. 16). The colour of
2
2
nanocrystal disappeared only in presence of HgCl , while in other cases the colour retained.
2
To study the reaction in more details, UV–Vis spectra of silver nanocrystals was taken in the
presence of different concentration of HgCl solution. In absence of HgCl silver nanocrystal
2
2
1
5

showed a characteristic peak at 425 nm. As the concentration of HgCl increased, the
2
absorbance of nanocrystals decreased with hypsochromic shift with an isosbestic point at 380
nm which clearly indicated equilibrium between the reactant and product during the entire
course of reaction showed in fig. 17 (Nowicka-Jankowska,1971; Kubista,1993).
Conclusions
The objective set for this study had been to prepare silver nanocrystals in a simple,
cost–effective and eco–friendly way, and in doing so we used orange extract (Citrus sinensis)
as both reducing and capping agent. We successfully characterized the synthesized silver
nanocrystals having unique shape using various physico-chemical processes. Several
chemical and biochemical aspect of the nanocrystals were studied. It shows strong affinity
towards Bovine Serum Albumin. It can penetrate in the eukaryotic cell and can destroy
2
+
cancer cells. These silver nanocrystals are able to sense Hg by colour change down to
micromolar range and catalyses the reduction of methylene blue by ascorbic acid.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
N. Siddiqui thankfully acknowledges the Maulana Azad National Fellowship for a senior
research fellowship. M. M. Islam was grateful to Department of Science and Technology,
Govt. of West Bengal for their financial support (Project No:756 (Sanc.)/ST/P/S&T/9G-
3
4/2013). S. Mukhopadhyay gratefully acknowledges partial financial help from the UPE II
fund of Jadavpur University sanctioned by University Grants Commission, New Delhi. The
authors also like to thank Prof. P. Karmakar and Dr. D. Laha of Jadavpur University,
Department of Life Science and Biotechnology for various physical measurements.
1
6

References
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Graphical Abstrac
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5

Figure 1. Colour change of orange extract and AgNO mixture during the nanocrystal
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formation.
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6

Figure 2. UV–Vis spectra of AgNO during formation of nanocrystals. Inset: Change of
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absorbance vs. time.
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Figure 3. Dynamic light scattering plot of silver nanocrystal
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8

Figure 4. FTIR spectrum of pure orange fruit dry extract (A) and after reacting with AgNO3
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