Enhanced catalytic and antibacterial activities of silver nanoparticles immobilized on poly(N-vinyl pyrrolidone)-grafted graphene oxide

Article abstract of DOI:10.1039/c5ra13286j

Poly(N-vinyl pyrrolidone) (PNVP)-grafted graphene oxide (GO) (GO-PNVP) has been synthesized using a GO-based macro-RAFT agent prepared via click reaction of alkyne-terminated RAFT agent (S)-2-(propynyl propionate)-(o-ethyl xanthate) and azide-functionalized GO (GO-N₃). FTIR, XPS, Raman, TGA and DSC studies confirmed its formation. Silver nanoparticles are then immobilized on GO-PNVP and GO via in situ reduction of silver nitrate in the presence and absence of glucose at 40 and 95 °C, respectively. FT-IR, UV-Vis, XRD, SEM and TGA studies supported the incorporation of silver (Ag) nanoparticles. Ag nanoparticles immobilized on GO-PNVP are small, spherical and narrowly distributed (homogenous, monodisperse) compared to GO. These nanocomposites are explored as catalysts for the reduction of p-nitrophenol into p-aminophenol and also as antibacterial agents towards Gram(+) S. aureus and Gram(-) E. coli bacteria. Ag nanoparticle immobilized GO-PNVP showed efficient catalytic activity and excellent reusability along with an excellent antibacterial activity. Hence, grafting of PNVP enhances the catalytic and antibacterial properties of GO.

Full text of DOI:10.1039/c5ra13286j

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Page 1 of 29
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Enhanced Catalytic and Antibacterial Activities of Silver Nanoparticles
Immobilized on Poly(N-vinyl pyrrolidone)-Grafted Graphene Oxide
a
b
a
a,c
Shikha Singh, Ravi Kumar Gundampati, Kheyanath Mitra, K. Ramesh,
b
c
*
a
Medicherla V. Jaganaddam, Nira Misra, Biswajit Ray
a
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi – 221005, India
b
Molecular Biology Unit, Institute of Medical Science, Banaras Hindu University, Varanasi – 221005,
India
c
School of Biomedical Engineering, Indian Institute of Technology (Banaras Hindu University),
Varanasiꢀ221005, India
*
Corresponding author. Email: biswajitray2003@yahoo.co.in
Abstract
Poly(Nꢀvinyl pyrrolidone) (PNVP)ꢀgrafted graphene oxide (GO) (GOꢀPNVP) has been
synthesized using a GOꢀbased macroꢀRAFT agent prepared via click reaction of alkyneꢀ
terminated RAFT agent (S)ꢀ2ꢀ(Propynyl propionate)ꢀ(Oꢀethyl xanthate) and azideꢀ
3
functionalized GO (GOꢀN ). FTIR, XPS, Raman, TGA and DSC studies confirmed its
formation. Silver nanoparticles are then immobilized on GOꢀPNVP and GO via in situ
o
reduction of silver nitrate in the presence and absence of glucose at 40 and 95 C,
respectively. FTꢀIR, UVꢀVis, XRD, SEM and TGA studies supported the incorporation of
silver (Ag) nanoparticles. Ag nanoparticles immobilized on GOꢀPNVP are small, spherical
and narrowly distributed (homogenous, monodisperse) compared to GO. These
nanocomposites are explored as catalysts for the reduction of pꢀnitrophenol into pꢀ
aminophenol and also as the antibacterial agents towards Gram(+) S. aureus and Gram(ꢀ) E.
coli bacteria. Ag nanoparticles immobilized GOꢀPNVP showed efficient catalytic activity and
excellent reusability along with an excellent antibacterial activity. Hence, grafting of PNVP
enhances the catalytic and antibacterial properties of GO.
1

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Introduction
Several reports have recently been focused on the immobilization of nanoparticles onto
suitable substrates in order to prevent their agglomeration or, to enhance their efficiency
through a proper interaction with the substrate. Such approach shows a promising application
in optical, electronics, optoelectronic, electrochemical nanodevices and catalysis etc.
1
2
3
4
Recently, silica, alumina, zeolites , different types of carbon materials etc are used as
substrate to immobilize nanoparticles. Morphology of nanomaterials, which varied from
substrate to substrate, is an important factor to influence their properties. Graphene and its
derivatives are excellent substrates for catalysis as these materials possess large surface area,
unique thermal and chemical stability, excellent mechanical strength, and electrical
5ꢀ7
conductivity. Out of these, graphene oxide (GO) has the reduction capability for metal
o
8
precursors even at 0 C, but, GO itself simultaneously reduces to partially reduced graphene
RG) which undergoes agglomeration; such agglomeration hampers or, obstructs the
(
formation and growth of nanoparticles.
Water soluble and biocompatible polymer poly(Nꢀvinyl pyrrolidone) (PNVP), used
widely in pharmaceutical, cosmetic industries etc., can regulate the size and shape of
nanoparticles during synthesis by exhibiting dual characters of reducing as well as stabilizing
9
10
11
agent (protecting agent). Guo et al. and Nandi et al. synthesized recently PNVP grafted
covalently on GO and used the same as humidity sensors and compatibilizer. Herein, we
report the synthesis of PNVPꢀgrafted GO as substrate material to immobilise the
nanoparticles in anticipation that it can regulate the size of nanoparticles together with the
enhancement of the dispersibilty of GO in polar solvents.
Out of all reported metal nanoparticles, silver nanoparticles received huge attention
12
13ꢀ15
because of highest electrical conductivity, antibacterial properties
and excellent
2

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16
catalytic properties. Therefore, the synthesis and immobilization of silver nanoparticles on
17ꢀ22
a suitable substrate have been largely focussed by a considerable number of researchers.
Silver nanoparticles, from a catalytic perspectives, are unique material for a number of
important processes in the chemical industries for e.g., the selective oxidation of alkenes and
2
3ꢀ24
alkanes
and the reduction of 4ꢀnitrophenols (4ꢀNP) to 4ꢀaminophenols (4ꢀAP) which is a
potent intermediate in the manufacturing of many analgesic and antipyretic drugs and
2
5ꢀ26
nonhazardous materials such as paracetamol, phenacetin etc.
Here, we report the synthesis of PNVPꢀgrafted GO (GOꢀPNVP) via click chemistry
and reversible additionꢀfragmentation chain transfer (RAFT) polymerization methods and
used the same as a substrate material for the inꢀsitu immobilization of Ag nanoparticles in the
presence or, absence of glucose as reducing agent. First, graphene xide (GO) is synthesized
2
7
using improved Hummers method. Then, its –OH functional group is converted into the
corresponding bromoꢀderivative (GOꢀBr) on reaction with 2ꢀbromopropionyl bromide. This
GOꢀBr is converted into its corresponding azideꢀderivative (GOꢀN ) on reaction with sodium
3
azide. GOꢀN3 is then reacted with alkyne terminated RAFT agent (S)ꢀ2ꢀ(Propynyl
propionate)ꢀ(Oꢀethyl xanthate) via click chemistry to prepare xanthate RAFT agentꢀ
functionalized GO (GOꢀX). This GOꢀX is used as macroꢀRAFT agent for the synthesis of
PNVPꢀgrafted GO (GOꢀPNVP) via the RAFT polymerizaion of NVP using AIBN as initiator
o
in DMF at 60 C. Thereafter, Ag nanoparticles are immobilized on GO and GOꢀPNVP
substrates via the inꢀsitu reduction of silver nitrate in the presence and absence of glucose at
o
4
0 and 95 C, respectively. Finally, we investigated the catalytic efficiency of these Ag
nanoparticlesꢀimmobilized GO/GOꢀPNVP substrates for the conversion of 4ꢀNP to 4ꢀAP in
the presence of sodium borohydride reducing agent. Moreover, antibacterial activities of
these are also investigated by determining growth rate and minimal inhibitory concentration
of a Gramꢀnegative bacterium E. coli and a Gramꢀpositive bacterium S. aureus. In addition,
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an enzymatic assay study is performed using E. coli as representative bacterial model for the
understanding of antiꢀbacterial activity.
Experimental section
Materials
αꢀBromopropionyl bromide (98%, Aldrich, USA), triethylamine (TEA) (99%, S.d fine
chemicals, India), sodium azide (99%, Loba Chemie, India), CuBr (98%, Aldrich, USA),
4
anhydrous MgSO (Loba Chemie, India), hexane (CDH, India), tetrahydrofuran (THF) (98%,
Loba Chemie, India), carbon disulphide (CS ) (95% Loba Chemie, India), propargyl alcohol
2
(
98%, Loba Chemie, India), glucose (Fisher Scientific, India), AgNO
India), diethyl ether (S.d.fine chemicals, India), NaHCO (S.d.fine chemicals, India),
ꢀnitrophenol (4ꢀNP) (S.d. fine chemicals, India) were used as received. Cuprous bromide
CuBr, 98%, Aldrich, Steinheim, Germany) was purified using a reported procedure and
3
(S.d.fine chemicals,
3
4
(
28
dried under vacuum. Nꢀvinylpyrrolidone (NVP, 99%, Aldrich, USA) was dried over
anhydrous calcium hydride and distilled under reduced pressure. 2,2ꞌꢀazobis(isobutyronitrile)
(
AIBN) (98%, Spectrochem, India) was recrystallized from methanol. Anhydrous dimethyl
formamide (DMF) (99%, Spectrochem, India) used after two step distillations: azeotropic
distillation with benzene followed by distillation over anhydrous MgSO . Dry ethanol
Saraya Distilliary, India) was prepared by leaving over CaO for overnight followed by
4
(
distillation over fresh CaO. Deionised water was prepared by redistillation of the double
distilled water in an all glass distillation apparatus. (S)ꢀ2ꢀ(Propynyl propionate)ꢀ(Oꢀethyl
2
9
xanthate) (X3) was synthesized following the method reported by our group. GO was
27
prepared using the improved Hummers method.
Characterization
FTꢀIR spectra were recorded using a PERKIN ELMER Spectrum version 10.03.05.
ꢀ1
instrument spectrometer in the range of 400ꢀ4000 cm . UV visible spectra were recorded on
4

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SHIMADZU UVꢀ1700 pharma spec. Raman spectra of GO and all nanocomposites were
+
taken by RENISHAWRM 1000 Raman spectrophotometer having Argonꢀion (Ag ) laser
source of 514.5 nm. Thermogravimetric analysis (TGA) was performed using PERKIN
o
ELMERꢀSTA 6000 thermogravemetric analyser in the temperature range from 30ꢀ600 C
o
with a heating rate of 20 C/min under N atmosphere. Differential scanning calorimeter
2
(DSC) measurement was performed using METTLER STAR SW 10.00 DSC instrument
o
under N
2
atmosphere at a heating rate of 20 C/min. The instrument was calibrated with
indium before use. The XPS measurement was made in a Ultra high vacuum AMICUS
photoelectron spectrometer equipped with MgKα Xꢀray as a primary excitation and a
KRATOS VISION 2 SOFTWARE. The curve fitting of high resolution spectra were
performed with combined GaussianꢀLorentzian functions. The morphology of PNVP grafted
GO was observed using Scanning Electron Microscope QUANTA 200F operated at an
acceleration voltage of 20 KV. Average size of nanoparticles was reported as the average of
the diameters of 50 numbers of particles observed in the SEM micrographs. The scattering
pattern of the powder samples was recorded in a XꢀRay diffractometer BRUKER ECO D8
ADVANCE. Samples were grinded well before measurement, and the diffractogram was
o
o
recorded using nickelꢀfiltered Cu tube, Kα radition with 2θ ranging from 5 to 80 . Zeta
0
potentials were measured using HORIBAꢀNanoparticles analyzer SZꢀ100, Japan at 25 C for
reference samples dispersed in deionised water (0.1mg/mL) after ultrasonication for 15 min.
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Scheme 1 Synthesis of poly(N-vinyl pyrrolidone)-grafted graphene oxide (GO-PNVP)
Preparation of bromo-functionalized graphene oxide (GO-Br)
27
GO (300 mg) (prepared using Improve Hummers method ) taken in a 500 mL round bottom
flask was vacuum dried for 2 h and purged with nitrogen. Anhydrous DMF (200 mL) were
then added to the flask and sonicated for 1 h to prepare a colloidal suspension of graphene
oxide. To it, TEA (6 mL, 0.04 mmol) was added. Then, αꢀbromopropionyl bromide (9 mL,
o
0
.08 mmol) was added dropwise to the above reaction mixture under stirring at 0 C. The
o
resulting mixture was stirred for 3 h at 0 C and then at room temperature 48 h under N
2
atmosphere. The solid was then separated from the final reaction mixture via filtration,
washed with chloroform, excess of deionised water and finally with acetone and then dried
under high vacuum for 24 h at room temperature.
Preparation of azide-functionalized graphene oxide (GO-N3)
3
GOꢀBr (250 mg) dispersed in anhydrous DMF (150 mL) via sonication. To it, NaN (231.5
o
mg, 3.56 mmol) were then added. The resulting mixture was stirred for 18 h at 70 C under
2
N atmosphere. The resultant mixture was then transferred to the deionised water with
vigorous stirring. The final product (GOꢀN ) was collected by centrifugation and washed
3
repeatedly with excess of deionised water and finally washed with acetone and then dried
under high vacuum for 24 h at room temperature.
6

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Preparation of RAFT-agent functionalized graphene oxide (GO-X) by click reaction
GOꢀN (200 mg) were added into the dry DMF (60 mL) and dispersed by sonication. To it,
3
CuBr (1.907 mg, 0.01 mmol) and DMF solution of alkyneꢀterminated raft agent X3 (79.344
mg, 0.342 mmol) were added. The resultant mixture was transferred to an oil bath maintained
o
at 90 C and stirred for 18 h. For workup, the resultant product was obtained by centrifugation
and washed with excess of DMF to remove unreacted alkyneꢀterminated RAFT agent
followed by washing with excess of methanol and water, finally with acetone. The obtained
o
black solid product (GOꢀxanthate, GOꢀX) was dried under high vacuum for 24 h at 40 C.
Preparation of poly(N-vinyl pyrrolidone)-grafted graphene oxide (GO-PNVP) by RAFT
polymerisation
GOꢀX (150 mg) was dispersed in dry DMF (50 mL) by sonication. To it, AIBN (5.615 mg,
0.0342 mmol) and NVP (1.8 mL, 17.1 mmol) were added. The resultant mixture was
o
transferred to an oil bath maintained at 60 C and kept at this temperature under stirring for
24 h . Resultant reaction mixture was dried under vacuum and dissolved in 2 mL THF and
precipitated from 300 mL hexane. This process of dissolution in THF and precipitation from
hexane was repeated twice. The final product was separated by centrifugation and dried under
o
vacuum at 40 C for 24 h.
In situ synthesis of GO-supported silver nanoparticles in the absence of glucose
(
GO-Ag 95)
GO (25 mg) was dispersed in 50 mL of water under sonication for 1 h. AgNO
.17 mmol, 75 wt % with respect to GO) was added to the above GO suspension. The
3
(18.75 mg,
0
dispersion containing GO and AgNO was stirred at room temperature for 60 min to ensure
3
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o
the homogenous mixing and then transferred to an oil bath maintained at 95 C for 20 h. The
obtained product was filtered and freezeꢀdried.
In situ synthesis of GO-PNVP supported silver nanoparticles in the absence of glucose
(
GO-PNVP-Ag 95)
GOꢀPNVP (15 mg) was dispersed in 25 mL of water under sonication for 10 min. AgNO
wt % with respect to GO, 11.25 mg, 0.10 mmol) was added to the above GOꢀPNVP
suspension. The dispersion containing GOꢀPNVP and AgNO was stirred at room
3
(75
3
temperature 60 min for homogenous mixing and transferred to an oil bath maintained at 95
o
C for 20 h. The obtained product was filtered and freezeꢀdried.
In situ synthesis of GO-supported silver nanoparticles in the presence of glucose
(GO-Ag 40)
GO (25 mg) was dispersed in 50 mL of water under sonication for 1 h. AgNO (75 wt% with
3
respect to GO, 18.75 mg, 0.17 mmol) was added to the above GO suspension. After the
complete dissolution of AgNO
3
, glucose (33.75mg, 0.18 mmol) was added to above
o
suspension and then kept under stirring at 40 C for 20 h. The obtained product was filtered
and freezeꢀdried.
In situ synthesis of GO-PNVP-supported silver nanoparticles in the presence of glucose
(
GO-PNVP-Ag 40)
GOꢀPNVP (15 mg) was dispersed in 25 mL of water under sonication for 10 min. AgNO
75 wt% with respect to GOꢀPNVP, 11.25 mg, 0.10 mmol) was added to the above GO
3
(
suspension. After the complete dissolution of AgNO , glucose (20.25mg, 0.10 mmol) was
3
8

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o
added to above suspension and then kept under stirring at 40 C for 20 h. The obtained
product was filtered and freezeꢀdried.
Catalytic studies
To a vial containing 3 mL of freshly prepared 10 mM NaBH solution, 25 µL (10 mM) of 4ꢀ
4
NP solution was added. This was followed by the addition of a specific amount of
immobilized Ag nanoparticles (best studied at 0.5 mg catalyst). The solution was thoroughly
mixed, followed by measurements of timeꢀdependent absorption at 400 nm. Each individual
catalytic study using nanocomposites was performed in quadruplicate.
Antibacterial studies
Growth curves of bacterial cells exposed to nanocomposites
To determine the growth curves of Gramꢀpositive Staphylococcus aureus (S. aureus) and
Gramꢀnegative Escherichia coli (E. coli) cells exposed to nanocomposite samples (at 5 µg/mL
6
concentration). The bacterial cell concentration was adjusted to 4 × 10 CFU/mL and incubated
in a shaking incubator up to 6 h at 37 ºC. Growth curves of bacterial cell cultures were
attained through measures of the optical density at 600 nm. All measurements were carried
out in triplicate.
Determination of minimum inhibitory concentration
The antiꢀbacterial activity of normal GOꢀPNVP, GOꢀAg 40 and GOꢀPNVPꢀAg 95 were
determined by using LB (Luria–Bertani; LB) broth as diluent for the bacterial strains.
Inoculates were prepared by suspending the overnight culture growth in sterile LB media.
The prepared samples were dispensed into 10 mL of a sterile 0.8 wt% saline water containing
6
about 10 cfu/mL of E. coli and S. aureus, and then shaken at 37 ºC. Twoꢀfold dilution of
reference sample and standard drug sample in the 96ꢀwell plates were prepared. The final
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volume in each well plate was 0.2 mL and was incubated at 37 ºC for 24 h. The microwell
plates were read at 590 nm using an ELISA reader to determine the MIC value. MIC was
defined as the lowest concentration of an antiꢀmicrobial studied compound that inhibits the
3
0
visible growth of a microorganism after overnight incubation.
Evaluation of anti-bacterial mechanism
The antiꢀbacterial effect of GO nanocomposites was determined by measuring the activity of
βꢀDꢀgalactosidase. A modified oꢀnitrophenolꢀβꢀDꢀgalactopyranoside (ONPG) test procedure
31ꢀ32
was used in this experiment.
Standard solution containing a cell suspension of 1 mL with
6
ꢀ1
a concentration of 10 CFU mL and 1 mL of ONPG solution with a concentration of 25 mM
were added simultaneously into 5.5 mL PBS buffer solutions (pH 7.4) and kept at 37 ºC
incubator for 15 min under shaking. Then, four different types of GO each of 100 µL (0.5µg
ꢀ1
mL ) were added into a 96ꢀwell plate and labelled separately. Then 100 µL of the above
standard solutions containing the cell suspensions and ONPG in PBS buffer were added in
the respective wells and labelled. At different time interval the changes of optical density at
420 nm (OD 420 nm) were measured by using a Multiꢀlabel Microplate Reader (Perkin
Elmer VICTOR 3). At the same time two control samples such as bacterial strain with ONPG
ꢀ1
and PNVP (0.5 µg mL ) with bacterial strain and PNVP were also measured.
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Results and discussion
Fig. 1 FTIR spectra of GO, PNVP and GOꢀPNVP
FTꢀIR spectra of GO, PNVP and GOꢀPNVP are shown in Fig. 1 Spectrum of GO shows the
ꢀ1
strongest band at 3438 cm corresponding to the OꢀH stretching vibrations (present in
carboxylic acid and phenolic functional groups) associated through hydrogen bonding.
ꢀ1
Moreover, the presence of peaks at 1097 and 1384 cm corresponding to CꢀO vibration and
ꢀ1
two another peaks at 1630 and 1726 cm corresponding to the asymmetric and symmetric
stretching COO vibrations of the carboxyl groups also support its formation. Apart from the
characteristic peaks of GO, the spectrum of GOꢀPNVP shows the characteristic peaks of
ꢀ1
PNVP and aromatic triazole ring: OH stretching of hydrogen bonded water at 3450 cm ,
ꢀ1
carbonyl stretching (heteroatom present in the ring of pyrolidone) at 3214 cm , intense
ꢀ1
2
symmetric CH stretching of the backbone chain of PNVP at 2927 cm , CꢀH stretching at
ꢀ1
ꢀ1
ꢀ1
2
857 cm , C=O stretching at 1709 cm , amide and CꢀN stretchings at 1640 cm , C=C
ꢀ1
ꢀ1
ꢀ1
aromatic ring vibration at 1580 cm , CH scissorings at 1465 cm and 1430 cm , CH
2
ꢀ1
ꢀ1
ꢀ
bending at 1365 cm , CH
2
twisting at 1216 cm , CꢀC pyrrolidone ring breathing at 1017 cm
1
ꢀ1
,
CꢀC of the backbone chain at 810 cm , and NꢀC=O bendings of the pyrrolidone ring at
ꢀ1
6
71 and 597 cm . All these characteristic peaks of PNVP confirmed the successful
3
3
functionalization of GO with PNVP.
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Fig. 2 XPS spectra of (a) C(1s) and (b) O(1s) region of GO and GOꢀPNVP.
XPS spectra of C (1s) and O (1s) region of GO and GOꢀPNVP are shown in Fig. 2(a)
and (b), respectively. The C (1s) signals of GOꢀPNVP is stronger than that of GO with a
shifting in its binding energy to lower side (284.1 eV) corresponding to the aliphatic
hydrocarbon (CꢀC, CꢀH) of PNVP backbone chain. But the O (1s) signal of GOꢀPNVP is
weaker with respect to that of GO. The C/O area ratio is increased in GOꢀPNVP owing to the
increase in the carbon skeleton. These observations reveal the successful functionalization of
PNVP on the GO.
3
Fig. 3 XPS spectra of (a) Br(3d) region of GO, GOꢀBr and GOꢀN (b) N (1s) region of GO,
GOꢀN , GOꢀX and GOꢀPNVP (c) S (2p) region of GOꢀN , GOꢀX and GOꢀPNVP.
3
3
XPS spectra of Br (3d) region GO, GOꢀBr and GOꢀPNVP are shown in Fig. 3 (a) Br ꢀ
terminated precursor GOꢀBr exhibits a strong peak at 70 eV which are assigned to Br (3d)
signal. Absence of this peak in GO and GOꢀN indicates the successful bromination of GO
3
with αꢀbromopropionyl bromide and replacement of bromine by azide. XPS spectra of N (1s)
region of GO, GOꢀN
3
, GOꢀX and GOꢀPNVP are shown in Fig. 3 (b) In case of
GOꢀ
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PNVP, the observed shifting to the higher binding energy side (401 eV) corresponds to –Nꢀ
3
4
3
C=O group of PNVP. XPS spectra of S (2p) region of GOꢀN , GOꢀX and GOꢀPNVP are
shown in Fig. 3 (c) Sulphur terminated precursor GOꢀX revealed a strong peak at 162.5 eV.
This peak is absent in case of GOꢀN . Thus, it reveals the presence of xanthate in the sample.
3
Curve fitting of XPS spectra are shown in Fig. S2 (Supporting Informations). Other
characterizations such as Raman, XRD, TGA and DSC also support its formation (Fig. S1,
S3 and S4, Supporting Informations). All these results indicate the successful PNVP
functionalization of GO.
Fig. 4 FTꢀIR spectra of GO, GOꢀAg 40 and GOꢀAg 95.
The FTꢀIR spectra of GO, GOꢀAg 40 and GOꢀAg 95 are shown in Fig. 4 After the
incorporation silver nanoparticles, the peaks position of the functional groups on GO stilled
ꢀ
1
remained unchanged but the peak associated with asymmetric (1726 cm ) COO vibration
ꢀ1
shown decrease in intensity whereas that of the symmetric (1630 cm ) COO vibration
increased. Such change in peak intensity may be because of the interaction between the Ag
nanoparticles and the oxygen containing functional group. This result was consistent with
35
previous report.
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Fig. 5 FTꢀIR spectra of GOꢀPNVP, GOꢀPNVPꢀAg 40 and GOꢀPNVPꢀAg 95.
The FTꢀIR spectra of GOꢀPNVP, GOꢀPNVPꢀAg 40 and GOꢀPNVPꢀAg 95 are shown
ꢀ1
in Fig. 5 The peak associated with C=O stretching at 1710 cm shows decrease in intensity
ꢀ1
while that of resonance peak of C=O, CꢀN stretching band at 1640 cm shows increase or
strengthened with no considerable change in peak position . Interestingly, the peak associated
ꢀ1
with CꢀN bending mode at 1090 cm in GOꢀPNVP shifts to high wave number in GOꢀ
PNVPꢀsupported silver nanoparticles samples. This may be due to the interaction of the lone
pair of nitrogen with Ag. Thus, Ag nanoparticles are preferentially stabilized via coordination
36
through the lone pair of nitrogen.
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Fig. 6 XRD spectra of (a) GO, GOꢀAg 40 and GOꢀAg 95 and (b) GOꢀPNVP, GOꢀPNVPꢀAg
0 and GOꢀPNVPꢀAg 95
4
To confirm the inclusion of Ag nanoparticles in GOꢀAg 40, GOꢀAg 95, GOꢀPNVPꢀ
Ag 40 and GOꢀPNVPꢀAg 95 samples, XRD spectra was carried out (Fig. 6). The XRD
spectra of these samples show the characteristic diffraction peak of Ag at 2θ values of about
o
3
8.1, 44.3, 64.5 and 77.5 corresponding to (111), (200), (200) and (311) crystallographic
planes of face centred cubic (fcc) lattice of metallic silver nanoparticles ( JCPDS No. 04ꢀ
783). In case of GOꢀPNVPꢀAg systems, the presence of a new sharp peak at 2θ values of
0
o
37
about 30.8 is also observed. Similar observation is also reported in the literature. Presently,
it is not clear to us. Therefore, XRD study confirms the incorporation of Ag nanoparticles in
GO/GOꢀPNVP substrates.
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Fig. 7 SEM images of (a) GO (b) GOꢀAg 40 (c) GOꢀAg 95 (d) GOꢀPNVP (e) GOꢀPNVPꢀ
Ag 40 and (f) GOꢀPNVPꢀAg 95.
To get the idea about the size and shape of Ag nanoparticles in GO/GOꢀPNVP
substrates SEM technique is used. Fig. 7 shows the scanning electron micrographs of the GO,
GOꢀAg 40, GOꢀAg 95, GOꢀPNVP, GOꢀPNVPꢀAg 40 and GOꢀPNVPꢀAg 95 samples. GO
and PNVP samples show clearly the absence of nanoparticles, whereas all the other systems
have Ag nanoparticles. The average size of Ag nanoparticles in GOꢀAg 40, GOꢀAg 95, GOꢀ
PNVPꢀAg 40 and GOꢀPNVPꢀAg 95 systems are 58, 102, 30 and 15 nm, respectively. The Ag
nanoparticles over GOꢀPNVP substrate are small, spherical and narrowly distributed
(homogenous, monodisperse), whereas, in case of GO as substrate, its shows the formation of
nonꢀhomogeneous and relatively larger nanoparticles. This result implies that the presence of
the grafted PNVP on GO plays an important role as stabilizer on the nucleation and growth of
silver nanoparticles.
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UVꢀVis spectra and TGA thermograms of GOꢀAg and GOꢀPNVP Ag systems also support
the incorporation of Ag nanoparticles in GO / GOꢀPNVP (Fig. S5 and S7, Supporting
Informations)
Catalytic studies
4ꢀaminophenol (4ꢀAP) is a well known organic compound, useful in many applications,
2
5ꢀ26
38
including analgesic and antipyretic drugs,
photographic developers,
corrosion
39
40
inhibitors and anticorrosion lubricants. So, the reduction of pꢀnitrophenol (4ꢀNP) by
NaBH in the presence of noble metal nanoparticles catalysts has been intensively
investigated.
ꢀAP using prepared four different catalysts: GOꢀAg 40, GOꢀAg 95, GOꢀPNVPꢀAg 40, and
GOꢀPNVPꢀAg 95. Under neutral or, acidic conditions, aqueous 4ꢀNP solution exhibits a
4
17ꢀ22
Here, we monitored the time dependent extent of the conversion of 4ꢀNP to
4
17
4
strong absorption peak at 317 nm. Upon the addition of NaBH , the alkalinity of the
solution increases and the absorption peak shifts to 400 nm. When GO is added into the
aqueous mixture of 4ꢀNP and NaBH , the peak remains unaltered with time. However, the
4
addition of a small amount of prepared catalysts (Agꢀnanoparticles immobilized substrates)
causes the rapid fading and ultimate bleaching of the yellow colour (absorption peak at 400
nm) with the appearance of the new peaks at 230 nm and 298 nm corresponding to 4ꢀAP.
These changes indicate the successful conversion of 4ꢀNP to 4ꢀAP. The whole reduction
process of each substrate was monitored by measuring the time dependant UVꢀvis absorption
spectra of the reaction mixture solution (Fig. S8, Supporting Informations).
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v
Fig. 8 Plots of (a) C
t o 0 t 0
/C and (b) ln (C /C ) (C = absorbance of 4ꢀNP at 400 nm at initial time,
C
t
= absorbance of 4ꢀNP at 400 nm at specific time interval t) vs. reaction time at room
o
temperature (27 C)
0 t 0
Almost linear correlation is observed in the plot between ln (C /C ) (C = absorbance
of 4ꢀNP at 400 nm at initial time, C
t
= absorbance of 4ꢀNP at 400 nm at specific time interval
o
t) and reaction time for all supported Ag catalyst systems at room temperature (27 C) [Fig.
8]. So, the reduction reaction is pseudo first order type as expected. The pseudo first order
o
ꢀ
rate constants (k) at 27 C calculated from the corresponding slopes (fitted) are 1.17, 3.6 x 10
3
ꢀ3
ꢀ2 ꢀ1
,
7.1 x 10 and 1.9 x 10 s for GOꢀAg 40, GOꢀAg 95, GOꢀPNVPꢀAg 40 and GOꢀPNVPꢀ
8
Ag 95 systems, respectively. The corresponding calculated TOFs (turn over frequency, the
number of reactant molecules that 1 g of catalyst can convert into product in the given
2
0
17
18
18
ꢀ1 ꢀ1
interval of time) are 1.50 x 10 , 5.0 x 10 , 1.0 x 10 , and 2.51 x 10 molecules g s ,
respectively. All the four catalysts exhibited different rates of reaction along with its
efficiency. These results clearly indicate that the catalytic properties of Ag nanoparticles are
influenced by the size of the nanoparticles and the nature of the substrate materials used for
immobilization. Out of all these supported catalysts, GOꢀAg 40 shows an excellent rate of
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reaction with high TOF compared to others. Though particle size of this system is ~58 nm,
but in situ synthesis of Agꢀ nanoparticles involving glucose as reducing agent led to partial
reduction of GO also, making it less defective and more aromatic like RG (reduced graphene)
22
type. Thus, the base substrate become more compatible with aromatic 4ꢀNP and adsorbed it
easily through πꢀπ stacking, making the reduction process much more facile for immobilized
8
,41
Ag nanoparticles.
Whereas for GOꢀPNVPꢀAg 95 and GOꢀPNVPꢀAg 40 systems, the
immobilizing surface of GOꢀPNVP is like GO (more defective and less aromatic) and
populated with grafted PNVP chains, which made it much less compatible with 4ꢀNP as πꢀ
stacking is very much hindered. So, here, GOꢀPNVPꢀAg 95 having smaller size (~ 15 nm)
and well distributed Ag nanoparticles with effectively larger surface area showed faster
reaction rate as compared to GOꢀPNVPꢀAg 40 system having distributed nanoparticles of
larger sized (~30 nm). GOꢀAg 95 system is the least reactive amongst the four. Here, GO
8
itself acted as reducing agent for Ag nanoparticle generation. The corresponding
nanoparticles are of much larger size (~102 nm) having much less surface to volume ratio
and much less abundant. Moreover, the base substrate remained more defective, less aromatic
and less compatible with 4ꢀNP.
Fig. 9 Recyclability of catalysts (GOꢀAg 40, GOꢀAg 95, GOꢀPNVPꢀAg 40, GOꢀPNVPꢀAg
95) for the conversion of 4ꢀnitroꢀphenol into 4ꢀaminoꢀphenol.
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We further investigated the recyclability of these catalysts (GOꢀAg 40, GOꢀAg 95,
GOꢀPNVPꢀAg 40, and GOꢀPNVPꢀAg 95). For this, we carried out a set of reactions, in
sequence, where the ratio of the amount of catalyst and the reactants were kept the same.
Once the reaction was completed (as observed by the disappearance of the characteristic
absorption peak of 4ꢀNP at 400 nm), the catalysts were recovered by decantation of the
product reaction mixture. This was followed by washing of the solid composite with
deionised water. Then, 3 mL of the same starting reaction mixture was added to the cuvette
containing the washed catalyst. The absorbance decay of 4ꢀNP with time was monitored as
before. The completion of the reaction was taken as the second cycle. Similarly, the total
number of cycles repeated was six using the same catalyst. The results for all catalysts are
shown in Fig. 9. It reveals that the catalytic activity is retained quite well in GOꢀPNVPꢀAg
system compared to GOꢀAg system. For example, in case of GOꢀAgꢀ40, the reaction was
completed in 2 s in the first cycle; but it took 3000 s to be completed in the fifth cycle. The
same reaction in case of GOꢀPNVPꢀAgꢀ40 took only 900 s in the fifth cycle. The apparent
difference between the successive cycles could be due to the loss of catalyst during washing
in between two cycles. The nanoparticles adhered to the surface of GOꢀPNVP have greater
affinity via the pyrrolidone group of PNVP system as compared to GO (which partially
reduced during the Ag nanoparticles synthesis) and prevent the leaching of Ag nanoparticles
during the course of reaction. From systematic survey of literature for the catalytic
conversion of 4ꢀNP into 4ꢀAP using NaBH as reducing agent, Agꢀnanoparticle loaded GOꢀ
4
PNVP emerges as an efficient, workable, easily recoverable and reusable Ag nanoparticle
catalyst with reasonably high TOF [Table S9 (Supporting information)]. Such supported Ag
catalyst will eventually be useful in lowering the production cost in industry.
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Antibacterial studies
The antibacterial activities of PNVP, GOꢀPNVP, GOꢀAgꢀ40 and GOꢀPNVPꢀAgꢀ95 were also
investigated by determining growth rate and minimal inhibitory concentration of a Gramꢀ
negative bacterium E. coli and a Gramꢀpositive bacterium S. aureus. To measure the growth
rate of bacteria, cells were cultured in LB broth with PNVP, GOꢀPNVP, GOꢀAgꢀ40 and GOꢀ
PNVPꢀAgꢀ95 and the growth was monitored by the measurement of increase in optical
density (OD) at 600 nm. The obtained reliable growth curves of E. coli and S. aureus are
shown in Fig. 10(a) and (b). Except PNVP all the other samples exhibit antibacterial
Fig. 10 Effect of PNVP, GOꢀPNVP, GOꢀAg 40 and GOꢀPNVPꢀAg 95 at 5µg/mL on the
6
growth of (a) E.coli and (b) S.aureus at 10 CFU/mL concentration.
activities for both Gramꢀpositive and Gramꢀnegative bacteria. Using 5 µg/mL concentration,
GOꢀPNVPꢀAg 95 completely inhibited the bacteria cell growth of both Gramꢀnegative E. coli
and Gramꢀpositive S. aureus after around 4 h. So, among all the samples studied, GOꢀPNVPꢀ
Agꢀ95 showed excellent antibacterial property. In order to obtain more comprehensive
antibacterial activities of these nanocomposites and their lowest concentrations which
completely inhibit bacterial cell growth, we measured the minimal inhibitory concentration
(MIC) of these nanocomposites against both bacteria species as shown in Table 1. The result
clearly indicates that the MIC values of Gramꢀnegative bacteria are lower
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Table 1. Summary of minimal inhibitory concentration (µg/mL) against Gramꢀnegative and
Gramꢀpositive bacteria
Bacterial strain
E.coli (Gram-)
GO-Ag 40
0.56
GO-PNVP
0.23
GO-PNVP-Ag 95
0.17
1.92
S. aureus (Gram +)
2.3
2.26
than those of Gramꢀpositive bacteria. The observed trend may be from the distinct structure
of cell wall between Gramꢀnegative and Gramꢀpositive bacteria. The cell wall of Gramꢀ
negative bacteria E. coli is composed of thin peptidoglycan (meshꢀlike polymer consisting of
sugars and amino acids) layer (7ꢀ8 nm) with an additional outer membrane; whereas, Gramꢀ
positive bacteria S. aureus contains a thick peptidoglycan layer (20ꢀ80 nm) at the outside of
the cell wall without any additional outer membrane. The thick peptidoglycan of Gramꢀ
positive bacteria may also include other components such as teichoic and lipoteichoic acids
and complex polysaccharides, which serve to act as chelating agents and protects against
antibacterial agents such as antibiotics, toxins, chemical, and degradative enzymes.
Moreover, the antibacterial activity of Agꢀnanoparticles incorporated in GOꢀPNVP substrates
is superior with respect the other related reports of graphene oxide and its derivatives over E.
coli and S. aureus as shown in Table-S10 (Supporting Informations).
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.
Fig. 11 Optical density (OD) curves indicating the absorption of oꢀnitrophenol (ONP) from
different well plates at different time periods
To understand the antiꢀbacterial effect of these nanocomposites in more details, a βꢀ
Dꢀgalactosidaseꢀbased enzymatic assay has been performed using E. coli as representative
bacterial model. βꢀDꢀgalactosidase is an exoglycosidase (hydrolase enzyme) which presents
both in the human body and microorganisms such as E. coli and exhibits hydrolyzing activity
for the βꢀDꢀglycosidic bond present in sugar (disaccharides), fat and glycosides. By the action
of antibacterial agent, βꢀDꢀgalactosidase will leak out from the dead bacteria cells (E. coli),
and eventually catalyze the hydrolysis of oꢀnitrophenolꢀβꢀDꢀgalactopyranoside (ONPG) in
the medium into oꢀnitrophenol (ONP) which has the specific absorption at 420 nm. Samples
were added into the cell suspension containing ONPG and the absorption at 420 nm was
measured at different time intervals. The optical density (OD) curves are shown in Fig. 11.
OD of the untreated E. coli in the control well without any antiꢀmicrobial agent remained
intact as there was no release of βꢀDꢀgalactosidase into the suspension to catalyze ONPG. E.
coli treated with PNVP also exhibited no significant absorbance changes at the measured
time period. This result indicates that the catalytic effect or, interaction of PNVP does not
promote the conversion of ONPG into ONP i.e., antibacterial activity. However, GOꢀPNVP,
GOꢀAg 40 and GOꢀPNVPꢀAg 95 nanocomposites significantly disrupt the cell membrane of
42
43,44
E. coli as absorption at 420 nm increases with time. Ag nanoparicles as well as GO
are
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reportedly efficient antibacterial agents. Generally, Ag nanoparticles show antibacterial
+
properties by inhibiting the respiratory chain of bacterial cell and via sustained release of Ag
+
inside the bacterial cell (environment of lower pH). These Ag ions stimulates the production
of reactive oxygen species (ROS) which eventually (i) destroy the bacterial cell, (ii)
inactivate the bacteria protein, and (iii) inhibit the bacteria DNA replication, and electron
42
transport. Whereas, GO sheets intertwine the pathogens and most likely block the cell
membranes by mechanical wrapping resulting local perturbation and damage. This leads to
disturbance in normal metabolism of cells by blocking water channels and the absorption of
nutrients along with ROS production like Ag nanoparticles. All these eventually lead to the
4
3,44
bacterial cell death.
Any system containing both GO and Ag nanoparticles should
expectedly show better antibacterial properties. Here, we have considered two composite
systems, GOꢀAg 40 and GOꢀPNVPꢀAg 95, which showed excellent catalytic properties.
Moreover, we have chosen GOꢀPNVP system to study the effect of the grafting of PNVP on
GO. Among these three GO based systems, antibacterial activity of GOꢀAg 40, although it
contains Ag nanoparticles, is inferior to that of GOꢀPNVP and GOꢀPNVPꢀAg 95. This may
be because of the superior cellꢀadhesion properties of PNVPꢀgrafted GO compared to GO
owing to the higher complexing ability of the grafted PNVP with the functional groups of cell
membrane and the higher dispersibility of the PNVPꢀgrafted GO in the medium. Indeed, zeta
potential study supports the higher dispersibility of GOꢀPNVP systems in deionized water
(Table S11, and Figure S12, Supporting informations). GOꢀPNVPꢀAg 95 shows relatively
better antibacterial properties over GOꢀPNVP obviously owing to the presence of Ag
nanoparticles. Therefore, grafting of PNVP on GO enhances its antibacterial properties.
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Conclusion
PNVPꢀgrafted GO has been synthesized using GOꢀbased macroꢀRAFT agent prepared via
click reaction of azideꢀfunctionalized GO and alkyneꢀterminated RAFT agent X3 and
characterized by FTIR, Raman, XPS, TGA and DSC studies. Ag nanoparticles are then
immobilized on the surface of GO and GOꢀPNVP. FTꢀIR, UVꢀVis, Raman, XPS, TGA,
DSC, and SEM studies support their formation. Variation of immobilizing substrate and
reaction condition leads to considerable difference in the morphology and distribution of Ag
nanoparticles. Nanopartciles immobilized on GOꢀPNVP substrate are small, narrowly
distributed compared to that on GO. Using these nanocomposites, the catalytic effect for the
reduction of pꢀnitrophenol to pꢀaminophenol, and the antibacterial properties towards
Gram(+) S. aureus and Gram(ꢀ) E. coli bacteria are explored. GOꢀPNVPꢀAg showed efficient
catalytic activity and excellent reusability for the reduction process. This nanocomposite also
showed an excellent antibacterial property compared to simple GO based composites. Hence,
we conclude that grafted PNVP enhances the properties of GO as immobilizing base for Ag
NP as the nanoparticles adhered to surface of GOꢀPNVP with greater affinity via the
pyrrolidone group of PNVP and make it much suitable system for catalytic and antimicrobial
application.
Acknowledgement
BR gratefully acknowledges the financial support of DST (Govt. of India) via DST Nano
Mission Project. SS and KM acknowledge UGC (Govt. of India) and Banaras Hindu
University for Research Fellowships. Authors acknowledge Prof. R. K Singh (Dept. of
Physics, BHU), Prof. A. S. K Sinha (Dept. of Chemical Engineering, IITꢀBHU) and Prof. P.
Maiti (School of Material Science and Technology, IITꢀBHU) for providing Raman, XPS and
Zeta potential measurement facilities, respectively.
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Notes and references
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