Received: 7 June 2016 Revised: 16 August 2016 Accepted: 21 August 2016

DOI 10.1002/jmr.2565

RESEARCH ARTICLE

Green synthesis of silver nanoparticles using Pongamia pinnata

seed: Characterization, antibacterial property, and
spectroscopic investigation of interaction with human serum
albumin
Maidul Beg1 | Anukul Maji1 | Amit Kumar Mandal2 | Somnath Das1 | Mt Nasima Aktara1 |

Pradeep K. Jha3 | Maidul Hossain1*

1
Department of Chemistry and Chemical
Technology, Vidyasagar University, Midnapore Abstract
721102 West Bengal, India In recent years, green synthesized nanoparticles from plant extract have drawn a great interest
2
Department of Microbiology, Vidyasagar due to their prospective nanomedicinal application. This study investigates a proficient, safer,
University, Midnapore 721102 West Bengal, and sustainable way for the preparation of AgNPs using medicinal plant Pongamia pinnata
India
3
(family: Leguminoseae, species: Pinnata) seeds extract without using any external reducing and
School of Medical Science and Technology,
stabilizing agent. Both ultraviolet‐visible spectrum at λmax = 439 nm and energy dispersive X‐ray
Indian Institute of Technology, Kharagpur
721302, India spectra proof the formation of AgNPs. An average diameter of the AgNPs was 16.4 nm as
Correspondence revealed from transmission electron microscope. Hydrodynamic size (d = ~19.6 nm) was deter-
Dr Maidul Hossain, Department of Chemistry mined by dynamic light scattering (DLS). Zeta potential of AgNPs was found to be −23.7 mV,
and Chemical Technology, Vidyasagar
which supports its dispersion and stability. Fourier transform infrared study revealed that the
University, Midnapore, 721102 West Bengal,
India. O ─ H, C ═ O, and C‐O‐C groups were responsible for the formation of AgNPs. The antibacterial
Email: hossainm@mail.vidyasagar.ac.in activity of the synthesized AgNPs was checked against Escherichia coli ATCC 25922. AgNPs at its
LD50 dose exhibited synergistic effect with ampicillin. Because protein‐AgNPs association
greatly affects its adsorption, distribution, and functionality and can also influence the functions
of biomolecules. So in order to understand the adsorption and bioavailability, we investigated by
fluorescence, ultraviolet‐visible, and circular dichroism spectroscopic methods the interaction of
synthesized AgNPs toward human serum albumin. The binding affinity and binding sites of
human serum albumin toward AgNPs were measured by using the fluorescence quenching data.
The circular dichroism spectroscopic results revealed that there was a negligible change of α‐
helical content in their native structure. Overall, these AgNPs show versatile biological activities
and may be applied in the field of nanomedicine.

K E Y W O RD S

antibacterial activity, binding affinity, conformational change, fluorescence quenching, FT‐IR, green
synthesis, HSA‐nanoparticle interaction, silver nanoparticles, TEM

1 | I N T RO D U CT I O N dispersities.1–7 But most of these techniques are extremely expensive

and also involve the necessity of toxic hazardous chemicals that may
Nanobiotechnology is a result of the intersection between adversely affect the biological systems and the environment also.
nanotechnology and biotechnology, which is an interesting area Hence, there is an earnest and insistent necessity of an inexpensive,
dedicated to improve the efficacy of nanoscale structures for highly substitute, and eco‐friendly technique for NP synthesis. The green
developed biotechnology. An important platform of research in this synthesis is the latest safer, faster, 1‐step, cost‐effective, nontoxic,
field is the synthesis of nanoparticles (NPs) by several methods with and eco‐friendly route for the NPs synthesis. It was reported earlier
various chemical compositions, shapes, sizes, and controlled that both biomolecules and living organisms could serve as a ready

J. Mol. Recognit. 2016; 1–8 wileyonlinelibrary.com/journal/jmr Copyright © 2016 John Wiley & Sons, Ltd. 1
2 BEG ET AL.

source of reducing and stabilizing agents for the synthesis of various 2.2 | Methods
nanomaterials.8,9 A survey of earlier literature suggests that the extract
2.2.1 | The preparation of Pongamia pinnata seed extract
of seeds and leaves of plants can be used for the synthesis of silver (Ag)
Fresh P. pinnata seeds were washed 4 to 5 times thoroughly with
and gold (Au) NPs.10–12 AgNPs were used in different area of medicine,
distilled water and cut into fine pieces and further sterilized the surface
industries, packaging, accessories, animal husbandry, cosmetics, mili-
with 90% ethanol. Subsequently, 15 g of these seeds were transferred
tary, and health. AgNPs exhibit potential antimicrobial effects on infec-
into 250‐mL sterilized Erlenmeyer flask and boiled at 90oC with
tious organisms such as Bacillus subtilis, Vibrio cholerae, Escherichia
150‐mL double distilled water for 45 minutes and then cooled to room
coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Syphilis
temperature. The greenish yellow color extract of seeds was filtered
typhus.13,14
through Whatman filter paper no. 41, and after sonication it was
In recent decades the application of NPs has been developed sub-
stored at room temperature for further study.
stantially. Inspite of this, there is very little knowledge related to the
15
various responses of biological systems when face up to the NPs.
2.2.2 | Synthesis of silver nanoparticles
Inside the living system, NPs may be covered by the protein and show
drastic effect on the conformation and functions of the protein.16 Five‐milliliter aqueous solution of 0.01‐M silver nitrate (AgNO3) was
There is no doubt about it that NPs can act as a double‐edged sword, mixed with 10 mL of the P. pinnata seed extract (PSE) under continu-
either as a platform for therapy or as a toxic agent depending on envi- ous stirring at 350 rpm. The consequent reduction of Ag+ ions was
ronment. This has led a keen attention to obtain the knowledge on monitored at room temperature. After 30 minutes of continuous stir-
protein‐NP interactions and its biological implications 17,18
to support ring, the color of the reaction mixture changed from greenish yellow
the controlled development of these promising materials. to yellowish brown and finally to dark brown, which was an indication
Human serum albumin is the most abundant protein in human blood of AgNPs formation. The reaction was continued further for 1 hour
plasma. It constitutes about half of serum protein. It transports until the completion of the reaction. Finally, the stable dark‐brown
hormones, fatty acids, and other compounds. It aids in maintaining the color of the reaction mixture indicated the completion of the reaction
buffer's pH and osmotic pressure, along with various other functions. (monitored by ultraviolet‐visible spectra).The AgNPs were purified by
There is a very few report on the fate of protein like human serum centrifugation at 13 500 rpm for 5 minutes at room temperature.
albumin (HSA)19 after its interaction with the green synthesized NPs. Supernatants were decanted and the precipitation of AgNPs was
On the other hand, Pongamia pinnata (which is found in many parts resuspended in deionized water .This process was repeated for 3
of the world) seeds are used for treatment of keloid tumors, hyperten- times. The resulting AgNPs were resuspended in deionized water and
sion, inflammations, pectoral diseases, chronic fevers, hemorrhoids and used for characterization.
anemia,20 skin ailments, and rheumatic arthritis.21,22 Seed powder is
used as a febrifuge, tonic in bronchitis, and whooping cough (CSIR, 2.2.3 | Estimation of AgNPs concentration
1948‐1998), in traditional medicine. The average diameter of almost spherical AgNPs was calculated from
Thus, the aim of the present investigation is to synthesize of new the transmission electron microscope (TEM) micrograph using Image
AgNPs by a faster, one‐step, and eco‐friendly approach using the J software. Molar extinction coefficient (ξmax) of the AgNPs was
seeds extract of medicinal important plant, Pongamia pinnata, which estimated by applying the (Mie theory‐based) power law,23 and
served as both reducing and stabilizing agent. The efficacy of AgNPs through the use of the Lambert‐Beer's law, the concentration of the
was studied against a Gram‐negative bacterium, E. coli ATCC 25922. AgNPs was determined.
Further, the synergistic antibacterial activity of P. pinnata seed extract
stabilized AgNPs at its LD50 dosage in combination with the antibiotic 2.2.4 | Instrumentations
ampicillin against the test strain, which were investigated, and the Ultraviolet‐visible spectra of the AgNPs and seeds extract were
results are reported. Moreover, this study focused on the interaction recorded by the Shimadzu UV‐1601 spectrophotometer (Shimadzu
of synthesized AgNPs with HSA by a spectroscopic technique to get, Corporation, Kyoto, Japan) operated at a resolution of 1 nm using
in more detail, insight of the bio‐physical binding mechanism and the quartz cuvette with 1‐cm optical path length and scanned from
conformational changes that can offer significant clues on the adsorp- 200‐ to 800‐nm wavelengths.
tion, biosafety, and bioavailability. The accurate molecular mass (m/z) of the compounds present in
the aqueous seeds extract of P. pinnata was determined by using
MALDI ToF Mass Spectrometer, VOYAGER‐DE PRO, Applied
Biosystems, California, USA.
2 | MATERIALS AND METHODS
The hydrodynamic radii and zeta (ζ) potentials of AgNPs were
measured by taking dynamic light scattering of the solution in Zetasizer
2.1 | Materials nano ZS instrument (Malvern Instrument, Malvern, Worcestershire,
Silver nitrate (99.999%) and HSA were purchased from Sigma‐Aldrich, UK) equipped with 532‐nm‐wavelength laser and 1‐cm optical path cell
Bangaluru, India. P. pinnata (Indian native name: karanja, Indian beech) using the process of laser Doppler electrophoresis at 25°C.
plant's seeds were collected from Srirampur (G.P‐1), Debra, West It was about 20 to 200 times that the diluted AgNPs solution was
Bengal, India. Prepared in the laboratory was 10‐mM citrate‐phosphate taken on the grids and dried in vacuum at room temperature for field
buffer of pH 7.10. emission scanning electron microscopic (FE‐SEM) analysis. FE‐SEM
BEG ET AL. 3

images of the samples were obtained by using SUPRA 40 Field intervals for determination of both minimum inhibitory concentrations
Emission Scanning Electron Microscope, Carl Zeiss SMT AG (MIC) and LD50 concentration (dosage that will cause a 50% reduction
(Oberkochen, Germany), operating at 20 kV. in bacterial population) of synthesized AgNPs by measuring optical
A droplet of aqueous monodispersed solution of synthesized density at λ600 nm. All the experiments were carried out in triplicate
AgNPs was spread on carbon‐coated copper grid (300 meshes) and and the mean value was reported.
was completely dried at room temperature for the TEM analysis.
TEM images were taken by means of an analytical TEM, FEI TECNAI 2.2.6 | Synergistic effect of AgNPs with ampicillin
G2 20S TWIN, at the accelerating voltage of 200 kV. The elements The additive or synergistic effect (if any) of the AgNPs was tested by
present in NPs were analyzed by energy dispersive X‐ray (EDX) using the protocol described earlier.26 Briefly, E. coli ATCC 25922
spectrometer. cells (of equal inoculums) were grown in flasks containing 10‐mL
Fourier transform infrared (FT‐IR) spectrum of AgNPs solution was MH broth supplemented singly with either 10‐μg/mL ampicillin or
monitored by using a Perkin Elmer Spectrum Two FT‐IR spectropho- 1‐nM (LD50 concentration) AgNPs, or both AgNPs (1 nM) and ampi-
tometer equipped with a zinc selenide (ZnSe) attenuated total reflec- cillin (10 μg/mL). A culture flask containing only inoculums and MH
tance accessory, LiTaO3 detector, and a KBr beam splitter at room broth, devoid of any antibiotics or NPs, was taken as negative
temperature (Perkin Elmer, Inc., 940 Winter St, Waltham, MA 02451, control. Growth was measured in terms of absorbance at λ600 nm.
USA), a resolution of 2 cm−1 and scanned in the range of 4000 to All the experiments were carried out in triplicate and the mean value
400 cm−1. The spectrum of seeds extract was also obtained in the was reported.
identical and instrumental conditions.
Fluorescence spectra of proteins (5‐μM HSA) were recorded by 2.2.7 | Study of AgNPs internalization
using a Perkin Elmer LS55 Fluorescence Spectrometer, USA.The emission AgNPs‐treated bacterial cells were fixed on a slide and were
scan was operated with the fixed slit width of 2.5 nm at 300 to 500 nm permeabilized with methanol as described earlier,25 stained with
range. The excitation wavelength was set at 295 nm for HSA to excite 4,6‐diamidino‐2‐phenylindole and finally subjected to flow cytometry
the tryptophan (Trp) residues selectively. Synchronous fluorescence analysis to validate internalization of AgNPs inside the bacterial cells
was measured in the excitation range of 220 to 340 nm keeping Δλ at if any. Flow cytometry analysis was also carried out with control
15 and 60 nm.24 Three‐dimensional fluorescence spectroscopy bacterial cells (cells without any AgNPs treatment).
experiments were performed by using initial wavelength at 200 nm and
continued up to 350 nm with an increment of 10 nm for each scan.
Circular dichroism (CD) spectra were recorded at 25oC on a PC 3 | RESULTS AND DISCUSSION
driven Jasco J815 spectropolarimeter (JASCO International Co. Ltd.,
Tokyo, Japan) attached with a Peltier controlled cell holder and tem- 3.1 | Characterization of seeds extract
perature controller, PFD 425 L/15, in rectangular quartz cuvettes
The aqueous PSE extract contains some compounds such as
0.1‐cm path length in the 190‐ to 260‐nm‐wavelength region. Each
pongaflavanol, tunictachalcone, glybanchalcone, pomgamol, and galac-
spectrum was averaged of 5 successive accumulations at a scan rate
toside.27 The characteristic peaks corresponding to the m/z (MALDI)
of 100 nm/min, keeping a bandwidth of 1.0 nm at a sensitivity of
value at 408.41, 405.635, 385.461, 293.511, and 411.63 were
200 mdeg, and the baseline was corrected and smoothed within
obtained from the MALDI ToF Mass Spectroscopic results of aqueous
permissible limits using the inbuilt software of the unit. A fixed concen-
PSE (Figure S1). The calculated molecular masses of pongaflavanol
tration of HSA (1μM) was titrated with increasing concentration of
(C26H32O4, 408.23), tunictachalcone (C26H30O4, 406), glybanchalcone
AgNPs. Ellipticity values were expressed in terms of the mean residue
(C22H25O6, 385), pomgamol (C18H14O4, 294), and galactoside
molar ellipticity [θ] in units of degree square centimeter per decimoles.
(C29H48O, 412) are in close agreement with the m/z (MALDI) value
Secondary structural calculations were performed by the software
of 408.41, 405.635, 385.461, 293.511, and 411.63, respectively.
supplied by Jasco.
On the other hand, FT‐IR spectrum (Figure 1B) of the untreated
PSE exhibited the peaks at 3550, 2869, 2310, 1612, and 1072 cm−1
respectively for O ─ H, C ─ H, C ═ C, C ═ O and C‐O‐C stretch, which
2.2.5 | Antibacterial activity of AgNPs
were the constituent parts of the above mentioned compounds.
Monodispersed suspensions of AgNPs at varying concentrations were
prepared using ultrasonic vibration (100 W, 30 kHz) for 10 minutes in
deionized water. E. coli ATCC 25922 cells were inoculated from freshly
3.2 | Characterization of nanoparticles
prepared slants into Mueller Hinton (MH) broth (M391; Himedia, The addition of aqueous P. pinnata seeds extract to aqueous silver
Mumbai, India). Inoculated were 10 μL of exponential grown cells in nitrate solution showed a color change from light greenish yellow to
freshly prepared 100‐mL MH broth impregnated with varied concen- dark brown because of the formation of AgNPs. The excitation of sur-
trations of AgNPs (ranging between 0.5 and 2.0 nM) as described ear- face plasmon resonance with the AgNPs was the reason of color
lier.25 MH broth with bacterial inoculum without any AgNPs was used change.28 The surface plasmon resonance of this reaction mixture gen-
as negative control. MH broth containing AgNPs without any bacterial erated an absorbance peak near 439 nm that was absent for pure
inoculum was used as positive control. All the flasks were then incu- aqueous seeds extract (Figure 1A), supported the formation of AgNPs.
bated at 37oC and the bacterial growth was monitored at various time Thus, it indicated the bio‐reduction of silver ions to metallic AgNPs.
4 BEG ET AL.

FIGURE 1 A, Ultraviolet‐visible spectra of

Pongamia pinnata seeds extract (black line)
and AgNPs solution (pink line). B, Fourier
transform infrared spectrum of the AgNPs
from 0.01 (M) AgNO3 (black line) and
P. pinnata seeds extract (red line). C, Trans-
mission electron microscope images of the
AgNPs. D, Particle size histograms of the
AgNPs determined from transmission electron
microscope images. E, Field emission scanning
electron microscopic micrographs of the
AgNPs. F, Energy dispersive X‐ray pattern of
the AgNPs

Fourier transform infrared spectrum was monitored to analyze the at 2.806 keV. Similar type of works29,30 confirmed that the peak of
role of possible functional groups of phytochemical contents present in optical absorption occurring at around 3 keV is responsible for the
the crude aqueous PSE for the reduction of Ag+ to Ag and its stability. absorption of metallic AgNPs due to surface plasmon resonance. Ka
The FT‐IR peak position of aqueous PSE was slightly shifted toward of metallic Ag occurring at 22.162 keV and Kb at 25.002 keV are due
the frequencies at 3432, 2866, 1614, and 1070 cm−1 after the AgNPs to the innermost electron emission. On the other hand, the peak
synthesis by using this extract (Figure 1B). Thus, it is proved that corresponding to Cu came from the Cu grid.
mainly the O ─ H, C ═ O, and C‐O‐C functional groups of phytochem-
ical constituents (pongaflavanol, tunicatachalcone, pongamol, galacto-
3.3 | Evaluation of antibacterial activity and
side, and glybanchalcone)27 in aqueous PSE were responsible for the
formation of AgNPs and its stability.
synergistic activity
The result of the DLS analysis indicated that the AgNPs were in Synthesized AgNPs showed antibacterial activity against the test
the range of 5 to 30 nm and its average particle size (d) was strain. MIC was determined as the lowest concentration at which no
~19.6 nm (Figure S2). visible growth of the test pathogens occurs. Among the various
The TEM micrographs of the AgNPs at 20‐nm scale are shown in concentrations of AgNPs, 2.0 nM proved to be MIC. Figure 2A
Figure 1C. The AgNPs were spherical in shapes having maximum size range showed that 1‐nM concentration of AgNPs caused 50% growth
within 5 to 30 nm with an average diameter of ~16.4 nm, which was reduction (in terms of O.D value) when compared with that of nega-
determined from the histogram of particle size distribution (Figure 1D). tive control and hence depicted as LD50 dose. It was noted that ampi-
The FE‐SEM micrographs of the AgNPs at 100‐nm scale are cillin singly did not exhibit much affect on the growth of E. coli ATCC
shown in Figure 1E. It was observed that the AgNPs were almost 25922 (Figure 2B). But when added along with the LD50 dose of NPs,
spherical in shape, having a size range within 5 to 30 nm. it showed >90% growth inhibition. This study established the syner-
The EDX measurement further confirmed and strengthened the gistic or combinational effects of NPs with ampicillin. Similar type of
formation of 100% metallic silver. Figure 1F showed that the elements observation was reported in our earlier publication where glucan
in AgNPs were chiefly composed of Ag. The spectrum of the NPs capped AgNPs showed a synergistic effect with ampicillin,
described that La of the metallic Ag was found at 2.984 keV and Lb azithromycin, kanamycin, and netilmicin.31
BEG ET AL. 5

FIGURE 3 A, Ultraviolet‐visible spectral titration of human serum

albumin (HSA) with different concentrations of AgNPs (0, 0.101,
FIGURE 2 A, Effect of varying concentration of AgNPs on the growth 0.405, 1.01, and 2.01 nM) in citrate phosphate buffer of pH 7.10 at
of Escherichia coli ATCC 25922 in Mueller Hinton broth. B, Synergistic 25oC. [HSA] = 6.4 μM (fixed). B, Fluorescence emission spectra of free
antibacterial effects of the AgNPs at 1 nM in combination with ampi- HSA and HSA with different concentrations of AgNPs (0, 0.0995,
cillin (10 μg/mL). C, Flow cytometry study for AgNPs internalization: (i) 0.20689, 0.399, 0.598, 0.794, 0.995, 1.193, and 1.39 nM) in citrate
bacterial cells without any AgNPs treatment and (ii) bacterial cells phosphate buffer of pH 7.10 at 25oC. [HSA] = 5 μM (fixed).
treated with AgNPs λex = 295 nm. (Inset B) Fractional fluorescence (F0/F) plots of HSA
with the various concentrations of AgNPs

3.4 | Evaluation of AgNPs internalization

spectra of HSA at 279 nm gradually decrease with no shift of maxima.
Results showed that bacterial cells after AgNPs treatment exhibited
According to the earlier work,32,33 the assessment of these spectra
the increase of the side scattering intensity compared with that of con-
showed a decrease in intensity without any shift in band position
trol, which is an indication of increment in granularity within the cells
that suggested the occurrence of HSA adsorption on the surface of
that supports our claim of internalization of NPs inside the bacterial
AgNPs only.
cells (Figure 2C). Similar type of observation was noted earlier, where
internalization of glucan capped AgNPs inside Klebsiella pneumoniae
3.5.2 | Fluorescence quenching study of HSA by AgNPs
YSI6A was demonstrated by using flow cytometry.25
and evaluation of binding parameters
The fluorescence spectrum of HSA mostly comes from the single Trp‐
214 residue that is situated in physiologically essential subdomain IIA
3.5 | Interaction study between AgNPs and HSA
ligand binding site and makes the fluorescence quenching study of
3.5.1 | Interaction study by absorption spectroscopy AgNPs‐HSA complexes much fascinating. The variation of the intrinsic
The interactions of HSA with AgNPs have been studied to appreciate fluorescence intensity of HSA indicates the adsorption of Trp residue
the conformation evolution of the HSA as well as binding on the sur- to the NPs surfaces. Figure 3B represents the fluorescence response
face of AgNPs. Figure 3A shows the spectral changes of HSA of HSA (5 μM) with the addition of increasing concentrations of AgNPs
(6.4 μM) with the increasing concentration of AgNPs. The absorbance (0‐1.39 nM) by excitation at 295 nm (to neglect the contribution from
6 BEG ET AL.

phenylalanine and Tyr residues). The fluorescence intensity was

quenched because of the addition of AgNPs without appreciable
changes (either blue shift or red shift) in the emission wavelength.
The nonexistence of shifting in the emission wavelength with a
decrease in intensity indicated that the protein adsorption on the
NPs surfaces caused fluorescence quenching effect due to close
proximity of the Trp residues on the NPs surfaces or formation of
AgNPs‐HSA complexes upon interaction. This quenching in fluores-
cence properties of proteins has been analyzed by applying the
Stern‐Volmer (SV) equation 1.34

F 0 =F ¼ 1 þ kq τ0 ½Q ¼ 1 þ K SV ½Q (1)

where F and F0 are the maximum fluorescence intensities in the pres-

ence and absence of quencher respectively, kq is the bimolecular
quenching rate constant, τ0 is the lifetime of the fluorophore in the
absence of quencher, KSV is the SV constant, and [Q] is the concentra-
tion of quencher. Figure 3B (Inset) shows the linear Stern‐Volmer plots,
F0/F vs [AgNPs], which is an indication of one type of quenching.
Analyzing according to Equation 1, the value of KSV for AgNPs‐HSA
complexation was found to be 3.884 ± 0.011 × 108 M‐1. The existence
of large NPs‐protein interaction was confirmed by the large values
(108‐1011 M−1) of the SV quenching constant (KSV) with the NP size
range (5‐100 nm).35 If τ0 of HSA = 5 × 10−9s34 for the Trp residues,
then kq of HSA = 7.768 × 1016 M−1/s is much higher than the value
of diffusion‐controlled quenching process 1010 M−1/s. This clearly
indicated that the quenching was initiated from complex formation,
but not from dynamic collision. In fact, it is observed that the data that
abide by the SV equation are not evidence for the presence of a
dynamic quenching process. The static quenching process can be used
to treat these characteristics and have been explained because of
production of nonfluorescent AgNP‐protein complexes. In these cases FIGURE 4 Synchronous fluorescence spectra of HSA: A, Δλ = 15 nm;
the values of the number of binding sites (n) and the binding constant B, Δλ = 60 nm. [HSA] = 5 μM; λex = 280 nm; [AgNPs] = 0, 0.0995,
0.20689, 0.399, 0.598, 0.794, 0.995, 1.193, 1.39, 1.488, 1.58, and
(K) can be determined by using the Equation 2.36
1.671 nM

log½ðF0 −F Þ=F  ¼ logK þ n log½Q (2)

fluorescence spectra of HSA gradually decreased without any shift in
maximum emission wavelength with increasing the concentration of
where F0 and F are the maximum fluorescence intensities in the
AgNPs at Δλ = 15 and 60 nm. These results established the occurrence
absence and presence of quencher, respectively, and [Q] is the concen-
of significant fluorescence quenching without producing almost any
tration of quencher. Figure S3 represents the plot of log [(F0 − F)/F] vs
log [Q]. The values of n and K were obtained from the slope and inter- considerable disturbance in the micro‐environment or polarity around
the Tyr and Trp residues of HSA.
cept of the straight line as 1.01 and 3.897 × 108 M−1, respectively,
which establishes the binding affinity of HSA toward the AgNPs sur-
faces. The value of n(1.01) indicated that the interaction between
3.7 | Three‐dimensional fluorescence spectroscopy
AgNPs and HSA was 1:1 interaction; similar types of results were
The contour maps and three‐dimensional spectra of the HSA−AgNPs
obtained by Ahmad et al.36
complexes are shown in Figure S4c,d, respectively. According to these
figures, peak a is the Rayleigh scattering peak (λex = λem), and peak b
3.6 | Measurement of conformational change by
is the second‐order scattering peak, which is mainly caused by the
Synchronous fluorescence
π‐π* transition of the characteristic polypeptide backbone structure
Synchronous fluorescence spectra were measured by scanning concur- C ═ O of HSA.37
rently the excitation and emission monochromators, which only In Figure S4a,b, peak 1 (λex = 280 nm, λem = 340 nm) is the fluo-
showed the Tyr and Trp residues of HSA for wavelength intervals rescence peak that mostly presents the characteristic spectra of the
(Δλ) 15 and 60 nm, respectively.24 In Figure 4A,B, at Δλ = 15 and Tyr and Trp residues due to excitation of free HSA at 280 nm that
60 nm, the maximum emission wavelength of the Tyr and Trp residues reveals the intrinsic fluorescence of these moieties.34 In contrast to
were 286 and 280 nm, respectively. Here, the intensity of synchronous peak 1, another fluorescence peak 2 (λex = 230 nm, λem = 340 nm)
BEG ET AL. 7

has the characteristic polypeptide backbone structure of HSA.38 The this study established a significant interaction between the AgNPs
results (Table S1) indicated the fluorescence quenching of peaks 1 and the HSA.
and 2 without any change in Stokes shift; that is, the microenviron-
ments of Tyr and Trp residues did not undergo significant alterations
4 | CO NC LUSIO NS
during the formation of the HSA‐AgNPs complexes, which was also
supported by synchronous fluorescence spectra. Moreover, this study
The present work has successfully demonstrated an eco‐friendly green
indicated that the synthesized AgNPs had no significant effect on the
approach for the synthesis of AgNPs by using aqueous medicinally
HSA structure.
important plant PSE. The synthesized AgNPs were systematically char-
acterized by ultraviolet‐visible, DLS, FE‐SEM, TEM, EDX, and FT‐IR.
3.8 | Circular dichroism spectroscopy These AgNPs exhibited strong antibacterial activity against E. coli
ATCC 25922. The fluorescence spectroscopic results showed the
Circular dichroism measurements were monitored on free HSA and
binding affinity of HSA toward AgNPs with almost no change in polar-
HSA‐AgNPs systems to verify further HSA‐AgNPs complexation and
ity around Trp and Tyr residues. Finally, the CD spectra permitted us to
clearly to understand how the secondary structure of proteins (HSA)
establish the occurrence of a negligible secondary structural modifica-
was affected by the AgNPs. Figure 5 shows the CD spectra of HSA
tion in HSA upon interaction with AgNPs without any change in its
in the absence and presence of AgNPs. The α‐helix, β‐sheet, and ran-
basic structure. Moreover, from this systematic and comprehensive
dom coil in the secondary structure of free HSA changed from
investigation, we believe that these green synthesized NPs will be
55.3%, 26.3%, and 18.4% to 51.8%, 28.3%, and 19.9%, respectively,
useful as safe in the bionanomedical arena.
at a 2‐μM concentration of AgNPs (Figure 5 and Table S2). But the
shape and position of peaks in CD spectra remained unchanged.
ACKNOWLEDGEMENTS
Hence, we can conclude that the secondary structure of the HSA
was unfolded to a slight extent and its basic structure was kept intact This work was financially supported by the Department of Science and
upon binding with synthesized AgNPs.39 Technology (DST) Science and Engineering Research Board (SERB),
Government of India, NO SB/FT/CS‐051/2013 (SERB/F/3694/
2015‐2016).
3.9 | Zeta potential measurement
Zeta potentials are directly related to the net charges on the surfaces RE FE RE NC ES
of the macromolecules or particles. 40
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