Biomed. Mater. 18 (2023) 065025 https://doi.org/10.

1088/1748-605X/ad0274

Biomedical Materials

PAPER

Synthesis of copper-reduced graphene oxide nanomaterials using

RECEIVED
14 June 2023
REVISED
glucose and study of its antibacterial and anticancer activities
18 September 2023
Anu Singh1,3,∗, Akanksha Gautam2,3, Sagarika Banerjee1, Awantika Singh1 and Hemant R Kushwaha1,2
ACCEPTED FOR PUBLICATION
11 October 2023 1
School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
2
PUBLISHED Special Centre for Systems Medicine, Jawaharlal Nehru University, New Delhi 110067, India
3
1 November 2023 Equal authorship.
∗
Authors to whom any correspondence should be addressed.
E-mail: classicsingh@gmail.com and Hemantkushwaha@jnu.ac.in

Keywords: antibacterial, anticancer, reduced graphene oxide, copper nanoparticles, glucose

Abstract
In this work, glucose-capped copper nanoparticles decorated reduced graphene oxide
nanomaterial are synthesized at 100 ◦ C and 200 ◦ C via chemical reduction method and studied for
their antibacterial and anticancer activities. Synthesized nanomaterials were characterized using
x-ray diffraction, Fourier-transform infrared, transmission electron microscope, and RAMAN. It is
observed in transmission electron microscopy and selected area electron diffraction studies that
copper nanoparticles deposited onto reduced graphene oxide are smaller than nanoparticles
generated in the absence of reduced graphene oxide. Also, the size of copper nanoparticles
synthesized at 200 ◦ C is smaller than at 100 ◦ C. Results suggest that Cu/Glu/rGO synthesized at
both temperatures showed significant antibacterial activity against Escherichia coli and Bacillus
anthracis, similarly, showed significant cell death in cancer cell lines [Cal33 and HCT-116 p53
(+/+)]. Interestingly, the nanomaterials were seen to be more effective against the cancer cell lines
harboring aggregating mutant p53. Tumors with aggregating mutants of p53 are difficult to treat
hence, Cu/Glu/rGO can be promising therapeutic agents against these difficult cancers. However,
the antibacterial and anticancer activity of Cu/Glu/rGO synthesized at 100 ◦ C where Cu2 O form is
obtained was found to be more effective compared to Cu/Glu/rGO synthesized at 200 ◦ C where Cu
form is obtained. Though fine-tuning of the material may be required for its commercial
applications.

Abbreviations ROS Reactive oxygen species

NaOH Sodium hydroxide
Cu/Glu/rGO Glucose-capped copper nanoparticles ZOI Zone of inhibition
decorated reduced graphene oxide PBS Phosphate buffer saline
Cu/Glu Glucose-capped copper nanoparticles FBS Fetal bovine serum
Cu/rGO Copper nanoparticle decorated MTT 3-(4,5-dimethyl-2-thiazolyl]-2,5-
reduced graphene oxide diphenyl-2H-tetrazolium
GO Graphene oxide bromide
rGO Reduced graphene oxide MIC Minimum inhibitory concentration
Cu Copper ATP Adenosine 5′ -triphosphate
Cu2 O Cuprous oxide OD Optical density
CuO Cupric oxide EDTA Ethylene diaminetetra acetic acid
Glu Glucose ELISA Enzyme-linked immune sorbent
PEG Polyethylene glycol assay
FTIR Fourier-transform infrared DMSO Dimethyl sulfoxide
SAED Selected area electron diffraction DMEM Dulbecco’s modified eagle’s medium
TEM Transmission electron microscopy NCCS National Center for Cell Science
XRD X-ray diffraction LB Medium Luria-Bertani medium

© 2023 IOP Publishing Ltd

Biomed. Mater. 18 (2023) 065025
1. Introduction
In 1974, Norio Taniguchi, a Japanese scientist coined
the term ‘Nanotechnology’, a science and engineer-
ing field that significantly impacts all aspects of life.
Generally, the term ‘Nano’ suggests anything that is of
the order 10−9 nm in length [1–3]. While of material
sciences this is further described as materials where at
least one dimension of a single unit should be sized
between 1 and 100 nm, referred to as ‘nanomaterials’
[1–4]. Researchers are working tremendously in syn-
thesizing novel nanomaterials to upgrade the stand-
ards in all areas of life. Nanomaterials’ synthesis meth-
ods are broadly divided into three categories: physical,
chemical, and biological. Different types of nanoma-
terials have been synthesized under each category e.g.,
organic nanomaterials [5], inorganic nanomaterials
[6], carbon nanomaterials [7], and composites/hy-
brid nanomaterials [8]. These methods allow the syn-
thesis of nanomaterials with the desired physiochem-
ical properties i.e. shapes, sizes, and functionalities
[3, 9].
It has been observed that chemical synthesis
approaches are preferred compared to physical and
biological approaches as these are cost-effective, offer
high versatility in surface chemistry, are easy to
functionalize, provides high yield, controlled shape
and size, good thermal stability, high uniformity,
and reduced dispersity [10]. However, nanomater-
ials generated chemically via employing hazardous
chemicals limit their application in the health sector
[11, 12]. This limitation can be overcome by work-
ing on certain approaches like the use of natural
products or biomolecules as reducing and/or cap-
ping agents for the synthesis of nanomaterials, and/or
adding less toxic materials as substrates for nanoma-
terial generation [13–15]. Specifically, for chemical
synthesis of metal-based nanomaterials addition of
natural products and less toxic substrates (carbon-
based materials), are found to reduce the agglomer-
ation of nanoparticles and enhance their stability and
biocompatibility [16, 17].
Among various metal-based nanomaterials,
researchers these days are focusing on exploring
copper-based nanomaterials for their biomedical
applications such as antibacterial [18] and antic-
ancerous agents [19]. Copper is a versatile metal
with several characteristics: low cost, easy availability,
high electrical conductivity, recyclability, and high
reactivity [20, 21]. But copper nanoparticles suffer
from high toxicity and agglomeration [21]. Recently,
various types of research have been conducted to
generate less toxic and stable copper nanomaterials
where biomolecules (such as glucose, starch, carbo-
hydrates, ascorbic acid, etc) have been incorporated
with and without substrates to control aggregation
and enhance stability and biocompatibility [22–24].
2
A Singh et al
One such substrate is graphene and its different forms
graphene oxide (GO) and reduced graphene oxide
(rGO) which have been utilized as a significant sub-
stitute or support material for different biomedical
applications [24]. Graphene is an allotrope of car-
bon as a solitary layer of particles in a 2D hexagonal
cross-section in which one molecule frames every
vertex. Graphene possesses remarkable properties
such as thermal stability, mechanical area, high con-
ductivity, large surface area, etc. [25, 26]. In GO,
sp2 -bonded carbon molecules are arranged in a sol-
itary, two-dimensional hexagonal shape and possess
oxygen-containing functional groups on the basal
plane (hydroxyl and epoxy) and edges (carbonyl,
carboxylic). While rGO has less oxygen content than
GO, it exhibits a large surface area, high reactivity,
and biocompatibility [22, 24, 27–29].
In the past few years, some of the research
groups have been working on utilizing graphene
and its derivatives to generate biocompatible cop-
per/copper oxide nanomaterials with significant anti-
bacterial activity. Yang et al reported the synthesis
of stable rGO-Cu2 O nanocomposites using ascorbic
acid in the presence of PEG and sodium hydrox-
ide at room temperature, synthesized nanocompos-
ite shows excellent antibacterial activities against
Escherichia coli and Staphylococcus aureus which were
maintained around 70% and 65% and were increased
by 40% and 35% compared with free Cu2 O after
immersing 30 d in PBS solutions [30]. Rajapaksha
et al 2019 reported GO-CuONPs nanocomposite
suggestively inhibiting the growth of both E. coli
and Salmonella typhimurium bacteria better than
CuO-NPs [31]. Similarly, Alayande et al reported
hydrothermally synthesized rGO-CuO nanocompos-
ite films that show good antibacterial activity with
complete bacterial inactivation compared to CuO
alone [32]. Xu et al synthesized CuO-rGO nanocom-
posite via a one-step hydrothermal method show-
ing 89% activity against E. coli and published the
outcomes on ROS generation via different doses
to Mouse fibroblast L929, showing the nanocom-
posites’ low cytotoxicity [33]. Chen et al reported
a green and economical route of rGO/Cu2 O syn-
thesis using cellulose acetate/CuCl2 for highly effi-
cient antibacterial killed 2.57-log E. coli cells after
3 h of treatment [34]. After antibacterial properties,
graphene-based copper/copper oxide nanomaterials
have also been studied for their anticancer properties
[26, 35]. Ismail et al synthesized copper/reduced
GO nanocomposite via sonochemical method using
honey, results show that H/Cu/RGO nanocomposite
has good antibacterial and cytotoxic activity against
cancer colorectal cell line (HCT11), even in low con-
centrations and concluded that this nanocomposite is
suitable for biomedical applications [36]. Kodous et al
used the ultrasonication method to produce Cu/RGO
Biomed. Mater. 18 (2023) 065025
nanocomposites that inhibited human breast cancer
cells MCF-7 cancer cells [37]. The significant antibac-
terial and anticancerous activities of these nanoma-
terials accredited to the synergistic effect of rGO and
copper/copper oxide nanoparticles routing different
mechanisms of action for killing bacterial and cancer
cells such as the production of ROS, cations release,
biomolecule damage, ATP depletion, disruption and
damage to the cell membranes [24, 27, 35].
These reported studies show successful synthesis
of GO/rGO and Cu-based nanomaterials, however,
the use of typical chemicals as reducing agents for syn-
thesis and the use of relatively high concentrations for
application studies (in most cases) limits their usage
in the healthcare sector. Considering all the above
facts, the present study aimed to synthesize rGO and
Cu-based nanomaterial using a partial green chem-
istry approach where glucose is used to synthesize less
toxic nanomaterial, additionally, high temperature is
used to generate small-size metal nanoparticles owing
greater efficiency.
The present study reports a temperature-
dependent synthesis of glucose-capped copper nan-
oparticles decorated with reduced graphene oxide
(Cu/Glu/rGO) at 100 ◦ C and 200 ◦ C via a chem-
ical reduction approach. Synthesized nanomater-
ial Cu/Glu/rGO has been compared with controls
i.e. synthesized rGO, synthesized glucose-capped
copper nanoparticles (Cu/Glu), and synthesized cop-
per nanoparticles decorated reduced graphene oxide
(Cu/rGO). All the nanomaterials have been charac-
terized using XRD, FTIR, TEM, and RAMAN. All
the nanomaterials have been studied for their anti-
bacterial and anticancer activity and compared. The
antibacterial studies have been carried out using the
Kirby–Bauer disk diffusion method for susceptibil-
ity testing and the broth dilution method for MIC
determination. Anticancer studies have been car-
ried out using MTT assay for cell viability. Results
obtained suggest that Cu/Glu/rGO possess significant
antibacterial and anticancer activity at low concentra-
tions, hence, this material is a promising alternative
to fight infectious diseases and cancers.
2. Materials and method
The biological activity of all the synthesized nano-
materials is tested against Gram-negative (E. coli)
and Gram-positive (Bacillus anthracis) bacteria.
Cell viability assays were performed using head
and neck squamous (Cal33) and human colon
colorectal HCT-116 p53 (+/+) cancer cell line
purchased from the NCCS, Pune. All the chem-
icals used were analytical grade i.e. copper nitrate
(1 M), glucose (1 M), sodium hydroxide NaOH
(1 M), pure graphite powder (99.99%), strepto-
mycin, penicillin, potassium bromide and MTT
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A Singh et al
reagent [3-(4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-
2H-tetrazolium bromide) were procured from
Sigma-Aldrich (USA). LB Medium, yeast extract,
peptone, sodium chloride (NaCl), agar powder,
sterile swab, DMEM, glutamine, PBS, trypsin EDTA
and sterile discs (DD036-1VL) were purchased from
HI-Media. FBS purchased from GIBCO, USA. DMSO
and ethanol were purchased from Merck. The 96-well
plates were purchased from a Thermofisher and cell
culture dishes from SPL life science.
Instruments used were analytical balance
(RADWAG), refrigerator (Samsung), pH meter
(TELEMODUAL), vortex mixture (VWR Scientific),
Ultrasonicator cleaner (L20560 Labman), hot air
oven (Techno), centrifuge (Biofuge), hot plate-
magnetic stirrer (MHPS 350) autoclaved, oven
(Thermo) incubator-shaker (Lab-Thermo), and
horizontal laminar airflow (Indigenous), and an
ultraviolet–visible spectrophotometer (Labman),
Humidified incubator (Thermo-IGISO) Elisa plate
reader (TECAN, sunrise), and vertical Laminar air-
flow (I-mest). All the glassware and plastic ware were
purchased from Borosil, India.
Modified Staudenmaier’s method [38] is used to
synthesize reduced graphene oxide (rGO) from nat-
ural bulk graphite powder [39]. Glucose-capped cop-
per nanoparticles (Cu/Glu), and glucose-capped cop-
per nanoparticles decorated reduced graphene oxide
(Cu/Glu/rGO) nanomaterials were synthesized by
chemical reduction method using glucose as an eco-
friendly reducing agent at different temperatures i.e.,
100 ◦ C and 200 ◦ C. Here, copper nitrate (1 M stock)
acts as a metal precursor, glucose (1 M) as a reducing
and capping agent (prevent oxidation of copper), and
NaOH (1 M) for pH regulation and rGO as substrate.
It is reported that copper salts can be reduced on heat-
ing above room temperature, therefore in all the nan-
omaterials synthesized for this work, the temperature
is also a major factor [40]. To understand the role of
glucose on the shape and size of copper nanoparticles
decorated reduced graphene oxide (Cu/rGO) in the
absence of glucose was also prepared at both temper-
atures. Dissolving 20 mg pellets of reduced graphene
oxide in 30 ml of distilled water provided a stock solu-
tion of 0.66 mg ml−1 of reduced graphene oxide. The
detailed procedure for the synthesis of nanomateri-
als at 100 ◦ C and 200 ◦ C is mentioned in the below
paragraph.
Cu/Glu: 52 ml of distilled water was taken in a con-
ical flask and placed on the hot plate with a mag-
netic stirrer at 300 rpm. The 4 ml of NaOH (1 M) was
added and stirred for 10 min then, 4 ml of glucose
(1 M) was added and stirred again for 10 min, a pale-
yellow color mixture obtained. Then, 100 µl of copper
nitrate (1 M) was added dropwise which generated a
light blue color, after continuous stirring for 20 min
the color changed from light blue to orange, and the
Biomed. Mater. 18 (2023) 065025
solution was continuously stirred for the next 20 min,
till the color changed to dark orange. The solution was
then centrifuged at 12 000 rpm for 20 min at 4 ◦ C,
the supernatant was discarded and the pellet was then
resuspended in 1 ml Milli-Q water and again centri-
fuged at 12 000 rpm for 20 min at 4 ◦ C. Afterward, the
supernatant was removed and the pellet was air-dried.
Cu/rGO: 52 ml of distilled water was taken in a conical
flask and placed on a hot plate with a magnetic stirrer
at 300 rpm. The 4 ml of NaOH (1 M) was added and
stirred continuously for 10 min. Then, 100 µl of cop-
per nitrate (1 M) and 200 µl rGO were added together
dropwise and stirred continuously for 20 min, a blue-
black color solution was obtained, and then again
stirred for the next 20 min, finally, generating a black
color solution. The resultant solution was centrifuged
at 12 000 rpm for 20 min at 4 ◦ C. The supernatant was
then discarded and the pellet was resuspended in 1 ml
Milli-Q water and again centrifuged at 12 000 rpm
for 20 min at 4 ◦ C. Afterward, the supernatant was
removed and the pellet was air-dried.
Cu//Glu/rGO: 52 ml of distilled water was taken in a
conical flask and placed on a hot plate with a magnetic
stirrer at 300 rpm. The 4 ml of NaOH (1 M) was added
and stirred continuously for 10 min, then, 4 ml of
glucose (1 M) was added and stirred continuously for
the next 10 min and a pale-yellow color solution was
obtained. Then, 100 µl of copper nitrate (1 M) and
200 µl rGO were added dropwise together, and a light
blue color solution appeared which slowly changed
from light blue to orange while stirred for 20 min,
the solution was again stirred continuously for the
next 20 min, the final solution of rust orange color
obtained. The resultant solution was then centrifuged
at 12 000 rpm for 20 min at 4 ◦ C, the supernatant
was discarded and the pellet was resuspended in Milli-
Q water which was then centrifuged at 12 000 rpm
for 20 min at 4 ◦ C. Afterward, the supernatant was
removed and the pellet was air-dried.
2.1. Characterization of nanomaterials
Powder XRD (PANalytical X’pert PRO) was used to
analyze the nature of phase and lattice parameters
of nanomaterials. FTIR spectrometer (PerkinElmer)
was used to identify the presence of functional groups
and chemical compounds. TEM (TECNAI-200Kv)
was used to observe the surface morphology, and size
of the synthesized nanomaterials (ultrasonically sus-
pended in 50% ethanol) at the electron accelerating
voltage of 200 kV. Raman spectroscopy (Varian FT-
Raman) demonstrated the chemical structure and the
presence of defects in the rGO. Raman spectroscopy
was used to determine the presence of carbon atoms
in the sample, especially considering the defects in
rGO nanosheets using solid state dewetting (SSD)
laser 532 nm wavelength and Raman spectrum mag-
nification recorded with the 100x objective.
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A Singh et al
2.2. Application studies
Air-dried pellets of rGO and all nanomaterials were
suspended in 50% ethanol and ultrasonically dis-
persed to form a stable dispersion or solution [41].
Stock solution (200 mg ml−1 ) of all the nanomaterials
was prepared for antibacterial studies and then work-
ing solutions (10, 50, and 100 mg ml−1 ) were pre-
pared from the same. Similarly, a stock of 1 mg ml−1
was prepared for anticancer studies, and working
solutions (25, 50, 100, and 200 µg ml−1 ) were pre-
pared from the same.
2.2.1. Antibacterial activity
Antibacterial activity studies of synthesized rGO
and nanomaterials against E. coli and B. anthracis
were performed using the Kirby–Bauer disk diffu-
sion method for susceptibility testing and broth dilu-
tion method for MIC determination [42]. Disk dif-
fusion susceptibility test is used to determine patho-
genic bacteria’s sensitivity or resistance against dif-
ferent nanomaterial concentrations. Similarly, MIC
assay is used to determine the lowest concentration
of nanomaterials that inhibits the visible growth of
bacterial cells. To determine the antibacterial activ-
ity of the nanomaterials, the bacterial reduction rate
(R %) was measured by counting the number of
colonies [33].
2.2.1.1. Kirby–Bauer disk diffusion method
Culturing-sub-culturing of both strains was per-
formed where a single colony was inoculated in
10 ml LB broth, separately, and incubated at
37 ◦ C overnight with 200 rpm agitation. Then, 1%
inoculum from this primary culture was inoculated in
10 ml fresh LB broth to obtain the secondary culture;
incubated at 37 ◦ C, 200 rpm, till the OD reached 0.3–
0.5 at 600 nm. Then, LB agar plates of both strains
were prepared separately via spreading (using sterile
swabs). Defined concentrations of synthesized nano-
composites were then loaded onto blank sterile discs
(6 mm diameter) which were then placed in an inver-
ted manner over spread plates and incubated for 24 h
at 37 ◦ C. The concentrations loaded onto sterile discs
were 20 µl of 10, 50, and 100 mg ml−1 of all synthes-
ized nanomaterials and rGO. This test was performed
in triplicate and the results were evaluated using the
ZOI in millimeters (including disc diameter).
2.2.1.2. Broth dilution method
To obtain the primary culture a single colony of both
bacterial strains was inoculated in nutrient broth, and
incubated overnight at 37 ◦ C with continuous shak-
ing at 200 rpm. Growth media for secondary cul-
ture was prepared in culture vials by adding differ-
ent concentrations (50, 100, and 200 µg ml−1 ) of syn-
thesized nanomaterials in 1 ml nutrient broth. Then,
1% of the primary culture of each bacterial strain
was transferred into these culture vials and incub-
ated at 37 ◦ C under continuous agitation at 200 rpm.
Biomed. Mater. 18 (2023) 065025
Bacterial growth was recorded at an interval of every
2 h by measuring the OD at 600 nm. As a comparison,
rGO was also studied under the same experimental
conditions.
To calculate bacterial reduction rate (R%) OD val-
ues of 4 h bacterial growth i.e. exponential phase was
used as follows (equation (1)),
Reduction rate (R%)
Control − Experiments
= ∗100. (1)
Control
2.2.2. Anticancer activity
The synthesized nanomaterials were tested for cell
viability against Cal33 and HCT 116 p53 (+/+) by
MTT assay. This colorimetric test is based on the
reduction of yellow tetrazolium salt of MTT reagent
to purple formazan product by mitochondrial dehyd-
rogenase in the metabolically active cells, reflecting
the metabolic rate of treated cells. Then, the crystals of
formazan are solubilized by adding DMSO. Then, the
number of viable cells is calculated by recording the
absorbance at 595 nm using an ELISA plate reader.
Initially, cell lines Cal33 and HCT 116 p53 (+/+)
received were cultured in DMEM supplemented with
5% FBS, glutamine, 1% streptomycin, and 1% peni-
cillin. Cells were grown in a cell culture plate, the pas-
sage is maintained in trypsin EDTA and incubated at
37 ◦ C in a humidified incubator of 5% CO2 . Then,
cells were seeded in a 96-well plate and incubated in
a 37 ◦ C incubator under humidified conditions for
24 h till the cells were more than 80% confluent. Post
incubation, fresh medium with different concentra-
tions of rGO and nanomaterials (25, 50, 100, and
200 µg ml−1 ) were added to the wells, separately. The
cells were again incubated for 24 h. Thereafter, 25 µl
(5 mg ml−1 in PBS) of MTT solution was added to
each well and incubated for 3–4 h in the dark. Then,
media was removed from the wells, and 100 µl of
DMSO was added to each well and incubated for 5–
10 min to solubilize the formazan crystals and form
a purple color. The OD was measured at 595 nm in
an ELISA multiwell plate reader. The OD was used
to calculate the percentage of cell viability using the
given formula (equation (2)). The experiment was
performed in triplicate to plot the graphs between the
percentage of cell viability and sample concentration
in µg ml−1 ,
cell viability = (OD value of sample
÷ OD value of control) × 100. (2)
3. Results and discussion
3.1. X-ray powder diffraction analysis
The XRD pattern obtained for rGO and nanomateri-
als is shown in figure 1 and the color of obtained pre-
cipitates is shown in the inset. Figure 1(a) shows two
5
A Singh et al
characteristic peaks at 24.52 and 43.16 2Θ brags angle
corresponding to (002) and (100) planes observed
for rGO [39]. XRD data for copper-based materials
shows the evolution of Cu, Cu2 O, and CuO forms
with crystalline peaks which can be accredited to
the compositions of the nanomaterials and synthesis
temperatures. The XRD reflection for the metallic
copper match with JCPDS File/Reference No: 98-004-
3493, Cu2 O JCPDS Reference No: 98-002-6963, and
CuO JCPDS Reference code 98-001-6025. Based on
XRD analysis, no impurities were found to produce
diffraction lines.
Figure 1(b) shows the XRD spectrum of Cu/Glu,
Cu/rGO, and Cu/Glu/rGO synthesized at 100 ◦ C.
XRD spectrum of Cu/Glu and Cu/Glu/rGO shows
the primary peak at 36◦ (111) and a secondary peak
at 43◦ (111) indicating the formation of Cu2 O and
Cu, respectively, while a clear peak obtained at 72◦
(222) confirms the formation of CuO (table 1). The
XRD spectrum of Cu/rGO shows the primary peak at
38◦ (002) and a secondary peak at 35◦ (200) indicat-
ing the formation of CuO. Small peaks for CuO and
two small peaks for Cu2 O were obtained while no
peaks for Cu (table 1). Figure 1(c) shows the XRD
spectrum of the nanomaterials synthesized at 200 ◦ C.
XRD spectra for Cu/Glu and Cu/Glu/rGO show the
primary peak at 43◦ (111) and a secondary peak at
51◦ (211) confirming the formation of Cu and CuO,
respectively, and a small peak at 36◦ (111) for Cu2 O
is obtained as described in table 1. The Spectrum for
Cu/rGO shows the primary peak at 38◦ (002) and a
secondary peak at 35◦ (200) indicating the presence of
CuO (table 1). Figures 1(b) and (c) shows the primary
peak of Cu2 O, secondary peaks of Cu, and the tertiary
peak of CuO obtained for Cu/Glu and Cu/Glu/rGO
synthesized at 100 ◦ C. The primary peak of Cu, sec-
ondary peaks of CuO, and tertiary peaks of Cu2 O
were obtained for Cu/Glu and Cu/Glu/rGO synthes-
ized at 200 ◦ C. This can be explained by exploring
the role of the temperature of nanomaterial synthesis
and the use of a capping agent. Results suggest that
high temperature increases the rate of reduction of
copper salts and the evolution of different forms of
copper (Cu2 O–Cu–CuO) takes place with the increas-
ing temperature [13, 16]. In Cu/Glu and Cu/Glu/rGO
synthesized at both temperatures, cubic phase Cu2 O
(rust orange) and Cu are predominantly formed due
to the presence of glucose as a capping agent, which
prevents further oxidation of copper [13, 43, 44]. It
is based on a reaction between reducing sugars (hav-
ing free aldehydes or ketones as functional groups)
heated in alkaline conditions and copper salt precurs-
ors that results in the change of color of the solution.
According to Benedict’s and Fehling’s tests, the solu-
tion changes color as the cupric ions (Cu+2 ) in the
solution are reduced to the insoluble cuprous ions
(Cu+ ), which precipitate out of the reaction [45, 46].
However, the nanocomposites synthesized at 200 ◦ C
Biomed. Mater. 18 (2023) 065025
Figure 1. XRD spectrum of (a) rGO (b) Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized at 100 ◦ C, and (c) Cu/Glu, Cu/rGO, and
Cu/Glu/rGO synthesized at 200 ◦ C.
were obtained in dark color as compared to 100 ◦ C
[47]. The net reaction between an aldehyde and the
CuII+ ions is shown in equation (3),
RCHO + 2 Cu2 + + 5 OH− → RCOO−
+ Cu2 O + 3 H2 O.
Cu/rGO shows black color precipitation at both
temperatures indicating that monoclinic cupric oxide
(CuO) form is predominant when the synthesis tem-
perature is more than room temperature and in the
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A Singh et al
absence of a reducing agent (equation (4)) [40, 48].
Due to this no difference in XRD spectrum was
observed for Cu/rGO synthesized at 100 ◦ C and
200 ◦ C,
(3) Cu (OH) (aq) + 2OH (aq) Cu(OH (aq) CuO (s)
+ 2OH (aq) + HO. (4)
3.2. Fourier-transform infrared (FTIR)
FTIR spectrum is used to identify the presence of
functional groups and chemical compounds [49].
Biomed. Mater. 18 (2023) 065025 A Singh et al

Table 1. XRD data showing different forms of copper obtained for Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized at 100 ◦ C and
200 ◦ C, respectively.

Temperature At 100 ◦ C At 200 ◦ C

Obtained peaks

Nanomaterials Cu Cu2 O CuO Cu Cu2 O CuO

Cu/Glu 43(111) b 36(111)a 77(222) 43(111) a 36(111)b 51(211)

74(220) 30(110) 74(220)
61(220)

Cu/rGO 61(220) 32(011) 61(220) 32(011)

73(311) 35(200)b 73(311) 35(200)b
38(002)a 38(002)a
48(−202) 48(−202)
53(020) 53(020)
58(202) 58(202)
66(−311) 66(−113)
68(022) 68(022)
75(−222) 75(−222)

Cu/Glu/rGO 43(111) b 36(111)a 77(222) 43(111) a 36(111)b 51(211)

74(220) 61(220) 74(220)
30(110)
a
depicts Primary peak.
b
depicts Secondary peak.

Figure 2 shows the FTIR spectra of rGO and
figures 2(a) and (b) shows the FTIR spectrum of
nanocomposites Cu/Glu, Cu/rGO, and Cu/Glu/rGO
synthesized at 100 ◦ C and 200 ◦ C. The details of
the peak position obtained with respect to functional
groups are shown in inset figure 2.
Cu/Glu and Cu/Glu/rGO synthesized at 100 ◦ C
show a peak at 633 cm−1 corresponding to CuI–O
stretching vibrations of Cu2 O form of copper nano-
particles as observed in XRD analysis [50]. Cu/rGO
synthesized at 100 ◦ C shows a peak at 498 cm−1
which corresponds to CuII–O stretching vibrations
of CuO form of copper nanoparticles as observed
in XRD analysis [50]. The different forms of cop-
per are obtained due to the synthesis of copper nan-
oparticles in the presence of glucose [confirmed by
carbonyl stretching in Cu/Glu (both aldehydes and
ketones) and in Cu/Glu/rGO (only ketones)] and
in the absence of glucose (Cu/rGO) (figures 2(a)
and (b)).
Cu/Glu and Cu/Glu/rGO synthesized at 200 ◦ C
show a peak at 620 cm−1 corresponding to Cu stretch-
ing vibrations of Cu form of copper nanoparticles
as observed in XRD analysis. Cu/rGO synthesized at
200 ◦ C shows a peak at 498 cm−1 which corresponds
to CuII–O stretching vibrations of CuO form of cop-
per nanoparticles as observed in XRD analysis [50].
As explained earlier, here the different forms of cop-
per are obtained due to the presence and absence of
glucose. However, the difference in copper forms in
Cu/Glu and Cu/Glu/rGO synthesized at 100 ◦ C and
200 ◦ C (i.e. Cu2 O and Cu) is obtained due to the
difference in synthesis temperature, it is also validat-
ing the XRD results.
Figures 2(a) and (b) show that the peak intens-
ity of the characteristic hydroxyl (O–H) group
of rGO is slightly decreased in Cu/rGO where
glucose is absent while no significant peak is
observed in Cu/Glu/rGO where glucose-capped nan-
oparticles are synthesized. Also, a slight difference
in the peak positions of carbonyl, aromatic, and
hydroxyl functional groups of rGO is observed in
Cu/rGO synthesized at both temperatures while
no significant peaks are seen in Cu/Glu/rGO
due to densely deposited glucose-capped copper
nanoparticles [49].
3.3. TEM analysis
The structural characterization of rGO (figure 3(a))
shows a wrinkled sheet and the crystallographic dif-
fraction pattern shows the formation of a ring struc-
ture (inset: SAED) [33, 51]. Figure 3(b) shows the
morphology of nanomaterials synthesized at 100 ◦ C
and 200 ◦ C with their respective SAED patterns. The
well-defined square shape copper nanoparticles are
observed for Cu/Glu nanomaterials synthesized at
100 ◦ C while irregular square shapes are observed for
nanomaterials synthesized at 200 ◦ C. Interestingly,
irregular spherical-shaped copper nanoparticles dec-
orated homogeneously on the rGO surface are
observed for Cu/Glu/rGO nanomaterials synthesized
at both temperatures [51]. TEM analysis for Cu/rGO
shows that rice-shaped copper nanoparticles are
deposited onto rGO sheets at both temperatures [52].

7
Biomed. Mater. 18 (2023) 065025
Figure 2. FTIR analysis of nanomaterials (a) rGO, Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized at 100 ◦ C, and (b) rGO,
Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized at 200 ◦ C.
SAED pattern of Cu/Glu, Cu/rGO, and Cu/Glu/rGO
synthesized at 100 ◦ C and 200 ◦ C is shown in
figure 3(b) (inset: SAED pattern). The bright spots
observed in all the samples clearly indicate the
formation of polycrystalline copper nanoparticles
[53, 54]. However, the diffraction ring observed
8
A Singh et al
in the background of Cu/rGO and Cu/Glu/rGO
samples is due to the presence of rGO which
is amorphous in nature. SAED patterns observed
for all synthesized nanomaterials confirm crystal-
line nanoparticle formation validating the XRD
data.
Biomed. Mater. 18 (2023) 065025 A Singh et al

Figure 3. TEM results (a) rGO (b) Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized at 100 ◦ C and 200 ◦ C, and (c) histograms
showing the average size range of synthesized copper nanoparticles. Inset: SAED pattern.

Further, the difference observed in the
morphology of the nanomaterials can be explained
in terms of their composition and temperature of
synthesis. The square and irregular sphere shapes
obtained in Cu/Glu and Cu/Glu/rGO can be credited
to the capping of copper nanoparticles by glucose
molecules which controlled their shape [55, 56]
as in Cu/rGO rice-shaped nanoparticles of copper
are formed where glucose was absent. Secondly,
the comparison between Cu/Glu and Cu/Glu/rGO

9
Biomed. Mater. 18 (2023) 065025
Figure 4. (a) RAMAN spectrum for rGO and Cu/rGO, Cu/Glu/rGO synthesized at 100 ◦ C and 200 ◦ C, respectively, and (b) peak
intensity ratio of nanomaterials.
nanomaterials shows that the addition of rGO as a
substrate prevents the agglomeration of copper nan-
oparticles and helps in forming smaller-size nano-
particles decorated homogeneously onto rGO sheets
[33]. The possible mechanism of the nucleation and
growth of small-size metal nanoparticles onto rGO
can be explained as follows: the nucleation strongly
depends on the structure of graphene form, espe-
cially on the number of oxygens containing functional
groups that act as a nucleation site. The high density
of oxygen moieties present in GO promotes more
nucleation than growth which results in a higher
number of created nanoparticles as compared to rGO
[57]. Similarly, rGO comprises more oxygen groups
as compared to pure metal nanoparticle synthesis
where rGO is not used. As a result, metal nano-
particles synthesized in Cu/Glu/rGO nanomaterial
have a smaller size and appear to have a larger specific
10
A Singh et al
surface area, which increases the probability of effect-
ive contact between nanomaterials and bacteria, thus
improving their antibacterial and anticancer proper-
ties. TEM images (figure 3(b)) and the histograms for
the size of nanoparticles (figure 3(c)) calculated with
the help of Image j and origin software clearly show
that the average size of the nanomaterials formed at
200 ◦ C is smaller as compared to the nanomater-
ials formed at 100 ◦ C [58]. This can be attributed
to the effect of temperature as the nanoparticle size
decreases with increasing temperature [17, 59].
3.4. RAMAN analysis
Raman spectroscopy is widely used to differentiate
crystal structures of carbon [60]. Figure 4(a) depicts
the Raman spectra obtained for rGO, Cu/GO, and
Cu/Glu/GO nanomaterials. The Raman spectra pat-
tern obtained for rGO shows two distinct strong
Biomed. Mater. 18 (2023) 065025
peaks at 1347 cm−1 (random arrangement of graph-
ite crystallites) called the D-band, and at 1583 cm−1
(stretching vibrational of C atoms in the sp2 hybrid-
ized plane) called the G band with 2D peaks obtained
around 2639 cm−1 with the peak intensity ratio
(I D /I G ) calculated to be 1.091 [60, 61].
The Raman spectra of Cu/rGO and Cu/Glu/rGO
synthesized at 100 ◦ C and 200 ◦ C show a slight
downshift of D and G peaks as compared to rGO
(figure 4(a)). This shift in peak positions towards
a lower wave number is attributed to the structural
modification occurring in the rGO sheets mediated
by interactions with copper nanoparticles during the
synthesis process [62, 63]. It is known that the higher
number of defects indicates a higher I D /I G ratio,
here, the I D /I G ratio for Cu/rGO and Cu/Glu/rGO
synthesized at 100 ◦ C and 200 ◦ C observed to be
less than the I D /I G ratio of rGO (figure 4(b)). The
lower intensity ratio illustrates that the defects of rGO
are healed during the deposition of glucose-capped
copper nanomaterials [64]. The 2D band (historic-
ally referred to as G) is an overtone of the D band
and can be related to the number of graphene layers
[65]. The 2D band of the separated GO nanosheets is
reported to be centered at 2683 cm−1 , which is typ-
ical for single-layer GO nanosheets [65–67]. In the
present study, the 2D band in rGO was obtained at
2693 cm−1 which is found to be shifted to 2683 cm−1
for Cu/rGO and 2680 cm−1 for Cu/Glu/rGO synthes-
ized at 100 ◦ C, respectively. Further, for Cu/rGO and
Cu/Glu/rGO synthesized at 200 ◦ C, the 2D band pos-
ition shifted to 2680 cm−1 as shown in figure 4(b).
This shift in 2D band position shows self-healing
characteristics of rGO due to the deposition of cop-
per nanoparticles which recovered defects generated
in the hexagonal network of carbon atoms [68].
4. Application studies
4.1. Antibacterial activity
4.1.1. Disc diffusion test
Figure 5(a) shows no growth takes place under the
rGO-loaded discs while no ZOI was formed around
the disc, stating that rGO can only kill the bacterial
cells that are in its close proximity via its nano-knife
mechanism and electron transfer mechanism [23, 69,
70]. The results obtained for antibacterial studies
of Cu/Glu, Cu/rGO, and Cu/Glu/rGO nanomaterials
synthesized at 100 ◦ C and 200 ◦ C are presented in
figure 5(b). The ZOI is clearly visible around the disc
loaded with 50 mg ml−1 and 100 mg ml−1 concentra-
tions of the nanomaterials confirming their inhibit-
ory antibacterial activity. However, weak activity was
recorded at 10 mg ml−1 concentration of all nano-
materials as mentioned in table 2. XRD data reveals
that Cu2 O, CuO, and Cu forms have been formed in
11
A Singh et al
these nanomaterials and it is known that pure cop-
per and copper oxides are capable of releasing ions
into the environment which can interact with bac-
terial cells and killing them via different mechanisms
[21, 24, 27]. Several studies reported that copper nan-
oparticles’ Cu2 O and CuO forms can release Cu+
and Cu+2 . The (cupric oxide) Cu+2 form of cop-
per significantly increases oxidative stress by produ-
cing ROS but the cuprous oxide Cu+ form can bind
to the intracellular protein much better than Cu+2 .
The results suggested that copper oxide nanoparticles
show better antibacterial activity than a pure form of
metal [21].
Generally, smaller-size nanoparticles have higher
antibacterial activity because they can easily penetrate
into bacterial membranes due to their large interfa-
cial area, thus enhancing their antibacterial efficiency
[23, 24]. However, some studies have shown that lar-
ger NPs are more effective, indicating that size alone
is not the most important factor of their toxicity
[71]. Similar findings are observed in this study where
large-size copper nanoparticles obtained in nanoma-
terials synthesized at 100 ◦ C showed better antibac-
terial activity compared to small-size copper nan-
oparticles obtained in nanomaterials synthesized at
200 ◦ C.
Table 2 shows that all the synthesized nanomater-
ials are more effective against E. coli than B. anthracis
as the positively charged copper ions can readily bind
to the negatively charged molecules on the bacterial
outer membrane, which disrupts the bacterial cell
membrane and cause membrane degradation, hence,
generating more oxidative stress (ROS) and killing
bacterial cells more effectively [24, 72]. Moreover,
100 mg ml−1 of Cu/Glu/rGO synthesized at 100 ◦ C
shows the best antibacterial activity compared to oth-
ers which can be accredited to the synergistic effects of
glucose, copper oxide, and rGO suggesting this nan-
omaterial to be a potential antibacterial agent [19, 22,
58]. It can be stated that graphene-based metal/metal
oxide nanoparticles involve various mechanisms to
kill bacteria through the physical direct interaction
of extremely sharp edges of nanomaterials with cell
wall membrane [18], ROS generation prevention of
bacterial proliferation and destruction of the integ-
rated cell membrane [73] even in dark [74], trap-
ping the bacteria within the aggregated nanomaterials
[75], oxidative stress [76], interruption in the glyco-
lysis process of the bacteria [77], DNA damage [78],
metal ion release [79] and recently reported contribu-
tion to the generation/explosion of nanobubbles [80].
4.1.2. Broth dilution method
MIC determination of synthesized nanomaterials
against E. coli and B. anthracis suggests that rGO
shows the least inhibitory effect whereas Cu/Glu,
Biomed. Mater. 18 (2023) 065025 A Singh et al

Figure 5. Antibacterial activity studies against E. coli and B. anthracis (a) rGO (10, 50, and 100 mg ml−1 ) and (b) nanomaterials
(10, 50, and 100 mg ml−1 ) synthesized at 100 ◦ C and 200 ◦ C.

Table 2. Zone of inhibition (mm) obtained against E. coli and B. anthracis.

ZOI (mm) for samples ZOI (mm) for samples

synthesized at 100 ◦ C synthesized at 200 ◦ C
Nanomaterials Concentration E. coli B. anthracis E. coli B. anthracis

Cu/Glu 10 mg ml−1 6 6 6 6
Cu/rGO 6 6 6 6
Cu/Glu/rGO 6 6 6 6

Cu/Glu 50 mg ml−1 8.33 ± 0.58 8 6.33 ± 0.58 6.33 ± 0.58

Cu/rGO 7.67 ± 0.58 6.67 ± 0.58 6 6
Cu/Glu/rGO 9 ± 1.00 8.67 ± 0.58 7 6.67 ± 0.58

Cu/Glu 100 mg ml−1 10 ± 1.00 9.33 ± 0.58 7 ± 1.00 6.33 ± 0.58

Cu/rGO 8 ± 0.58 7.33 ± 0.58 6.33 ± 0.58 6
Cu/Glu/rGO 12 10.33 ± 0.58 7.67 ± 0.58 7 ± 1.00
± Standard deviation (SD).

Cu/rGO, and Cu/Glu/rGO nanomaterials synthesized
at 100 ◦ C and 200 ◦ C inhibited bacterial growth
to varying degrees with increased concentrations
(figure 6). However, nanomaterials synthesized at
100 ◦ C retain better efficiency in killing bacterial cells
compared to nanomaterials synthesized at 200 ◦ C as
explained in the above section. It is also seen that
all the synthesized nanomaterials are more effect-
ive against E. coli than B. anthracis as positively
charged nanoparticles are more attracted toward
the negatively charged membrane of bacterial cells
[72]. It is observed that Cu/Glu and Cu/Glu/rGO
show a significant inhibitory effect as compared to
Cu/rGO for both temperatures attesting to the role
of glucose as explained above. However, Cu/Glu/rGO
shows the best results compared to other nanoma-
terials at the lowest concentration also credited to
the synergistic effect of copper, glucose, and rGO.
The values obtained for the bacterial reduction rate
(R%) at 4 h bacterial growth, revealed that R% is

12
Biomed. Mater. 18 (2023) 065025 A Singh et al

Figure 6. Growth curve study of E. coli and B. anthracis cultured at different concentrations of (a) rGO and (b) nanomaterials
synthesized at 100 ◦ C and 200 ◦ C, and (c) determination of bacterial reduction rate (R%).

increasing with increasing concentration of nanoma-
terials. The comparative study of synthesized nano-
materials with recently reported literature (table 3)
indicates that Cu/Glu/rGO is a promising antibac-
terial agent. Overall antibacterial results suggest that
Cu/Glu/rGO synthesized at 100 ◦ C has the poten-
tial to be used as an effective antibacterial agent.
This can be attributed to the different mechanisms of
killing bacterial cells such as the production of ROS,
cations release, biomolecule damages, ATP depletion,
and membrane interaction caused by copper nano-
particles [Cu2 O form] and membrane stress against
bacteria while contact with the sharp corner basal
planes of rGO, which results in disruption and dam-
age the cell membranes [23, 24]. The fine edges of
graphene sheets act as nano knives, cutting the bac-
terial cell membrane, which leads to the exposure of
intracellular components followed by cell death [22].

13
 Table 3. Comparative study of antibacterial activity of synthesized nanomaterials with recently reported literature.

S. No Sample Synthesis method Conc. of material Tested organism Testing method Inhibition % Mode of actions Ref.

1. GO–CuS Facile synthesis process at RT — E. coli/B. subtilis Plate count method 90%–100% Releasing Cu+ ions [81]

2. CuO-rGO Hydrothermal method 240 µg ml−1 E. coli MIC study 89% Synergistic effect of rGO and copper ions [28]

3. Cu2 O Wet chemical method 40 µg ml−1 /30 d E. coli/S. aureus Plate count method 40/35% Releasing Cu+ ions [30]
rGO-Cu2 O Wet chemical method 40 µg ml−1 E. coli/S. aureus Plate count method 70/65% Destroy the bacterial cells, copper ions react with
cytoplasmic constituents and finally inactivate the
bacteria.

4 CuO-rGO Hydrothermal method 240 µg ml−1 E. coli Growth curve study 100% ROS generation [23]
Biomed. Mater. 18 (2023) 065025

5. CuONPs-GO Thermal chemical reactions 1 mg ml−1 /3 h S. typhimurium/E. coli Plate count method ∼84%/72% ROS induces cell lysis [31]
2 mg ml−1 /3 h ∼98%/80%
3 mg ml−1 /3 h ∼90%/99%

6. rGO Hydrothermal method 4h E. coli Growth curve study ∼30% Oxidative stress via generating ROS [34]
6h ∼40%
8h 60%

14
rGO/Cu2 O 4h 70%
6h 92%
8h ∼1%
rGO/Cu2 O 20 µg ml−1 /3 h 0.67-log
40 µg ml−1 /3 h 1.17-log
80 µg ml−1 /3 h 2.57-log

7. CuO2 Two-step synthesis methods 6 µg ml−1 E. coli/S. aureus/MRSA Growth curve study — rGO/CuO2 nano-shuttles with the controllable release [80]
GO/CuO2 12.5 µg ml−1 of oxygen nanobubbles promoting interruption of
rGO/CuO2 25 µg ml−1 bacterial respiration.
rGO/CuO2 + laser 50 µg ml−1
100 µg ml−1

8. rGO Modified Staudenmaier’s 50 µg ml−1 E. coli & B. anthracis Growth curve study 54 46 Generating ROS via oxidative stress Present work
method 100 µg ml−1 74 72
200 µg ml−1 75 71
Cu/Glu/rGO Chemical reduction method 50 µg ml−1 E. coli & B. anthracis 92 91 Synergistic effect of rGO and release of copper ions,
synthesized at using glucose 100 µg ml−1 97 97 which kill the bacterial cells via generating ROS
100 ◦ C 200 µg ml−1 98 98
Cu/Glu/rGO 50 µg ml−1 E. coli & B. anthracis 91 91
synthesized at 100 µg ml−1 95 96
200 ◦ C 200 µg ml−1 98 98
A Singh et al
Biomed. Mater. 18 (2023) 065025
Figure 7. MTT assay showing nanomaterials percentage of cell viability against (a) Cal33 and (b) HCT-116 p53 (+/+) cells at
different concentrations.
4.2. Anticancer activity
4.2.1. Cell viability test
The findings of the present study indicated that the
cell viability under the treatment with rGO decreased
gradually for Cal33 cells as compared to untreated
cells, however, cell viability for HCT-116 p53 (+/+)
cells did not decrease much at the same concentra-
tions of rGO figure 7. It is observed that for both cell
lines, all the synthesized nanomaterials have more sig-
nificant anticancer activity than rGO. The Bar dia-
gram for both the cancer cell lines shows that Cu/Glu
and Cu/Glu/rGO show good anticancer activity com-
pared to Cu/rGO, though, nanomaterials synthesized
at 100 ◦ C show better efficiency concerning nano-
materials synthesized at 200 ◦ C. Figure 7 indicates
that the anticancer activity is dose-dependent i.e. the
viability of cells decreases with increasing concen-
15
A Singh et al
trations of rGO and all the nanomaterials. However,
all the nanocomposites’ more stringent anticancer
activity towards Cal33 is observed compared to the
HCT-116 p53 (+/+). It should be noted that while
HCT-116 p53 (+/+) cells harbor wild-type p53, Cal
33 cells harbor p53 aggregating mutant R175H) and
it is known that tumors harboring aggregating p53
mutants are difficult to treat as it is difficult to induce
cell death in cells with aggregating p53 mutation [82,
83]. Results obtained here indicate improved cyto-
toxicity in Cal33 cells with an endogenous R175H
mutation (aggregating mutant), these results can be
of higher value for future therapeutic approaches in
the treatment of aggressive and resistant tumors with
aggregating p53 mutations. Dose-dependent antican-
cer activity of all the synthesized nanocomposites is
mentioned in table 4.
Biomed. Mater. 18 (2023) 065025 A Singh et al

Table 4. Percentage of cell viability of Cal33 and HCT-116 p53 (+/+) obtained against rGO and synthesized nanomaterials.

Percentage of cell viability (%)

Samples CAL33 HCT-116 p53 (+/+)

Untreated
cells 100 100
−1 −1 −1 −1 −1 −1
Nanomaterials 25 µg ml 50 µg ml 100 µg ml 200 µg ml 25 µg ml 50 µg ml 100 µg ml−1 200 µg ml−1

rGO 75 68 65 21 90 74 72 38
Cu/Glu 66 55 27 4 68 59 38 4
(100 ◦ C)
Cu/GO 69 65 50 4 80 70 55 4
(100 ◦ C)
Cu/Glu/GO 62 50 14 3 64 50 32 3
(100 ◦ C)
Cu/Glu 68 60 29 4 70 65 42 4
(200 ◦ C)
Cu/GO 71 65 52 4 83 72 56 4
(200 ◦ C)
Cu/Glu/GO 66 52 21 3 66 55 36 4
(200 ◦ C)

Overall results of the MTT assay suggest that
Cu/Glu/rGO synthesized at 100 ◦ C has higher anti-
cancer activity. A comparative study of Cu/Glu/rGO
synthesized at 100 ◦ C with recently reported liter-
ature is shown in table 5. Graphene-based metal/
metal oxide nanocomposites show remarkable anti-
cancer activity as compared to pure rGO or metal
ions due to their synergistic effects and the death
of cancer cells occurring because of the produc-
tion of free radicals, thereby hindering their uncon-
trolled proliferation. Graphene-based nanomaterials
are cytotoxic because they damage cell membranes
due to their very sharp edges [84]. Besides alter-
ing cell morphology, nanocomposites decrease meta-
bolic activity in cells, increase oxidative stress, dam-
age mitochondria, and ultimately cause DNA damage
[35, 36, 84, 85]. Cu/Glu/rGO shows the synergistic
effect of copper, glucose, and reduced graphene
oxide.
The results obtained in the application stud-
ies of antibacterial and anticancer activity show
that Cu/Glu/rGO has more effect as compared
to all other synthesized nanomaterials. Further,
Cu/Glu/rGO synthesized at 100 ◦ C presents the
highest antibacterial and anticancer activity com-
pared to Cu/Glu/rGO synthesized at 200 ◦ C, sug-
gestive of better antibacterial and anticancer activity
offered by the Cu2 O form of copper along with the
synergistic effects of rGO and glucose as shown in
figure 8.

16
 Table 5. Dose and size-dependent comparative anticancer studies synthesized nanomaterials with recently reported literature.

Loaded volume (µg ml−1 )

S.no Sample Testing methods Size (nm) and exposure time Cell line Cell viability (%) References

1. GO-CuO MTT assay Agglomerated cluster 0 µg ml−1 /24 h HCT-116 cells 100% [86]
25 µg ml−1 /24 h 92.42 ± 2.0
50 µg ml−1 /24 h 61.81 ± 2.4
75 µg ml−1 /24 h 46.33 ± 2.0
100 µg ml−1 /24 h 31.81 ± 2.3

2. RGO/Cu3 0 MTT assay 50–110 nm 25 µg ml−1 /24 h MCF-7 cells 74.7 ± 1.24 [37]
50 µg ml−1 /24 h 58.9 ± 1.27
100 µg ml−1 /24 h 47.0 ± 1.56
Biomed. Mater. 18 (2023) 065025

200 µg ml−1 /24 h 35.5 ± 1.62

400 µg ml−1 /24 h 21.7 ± 1.43
800 µg ml−1 /24 h 10.2 ± 1.25
1600 µg ml−1 /24 h 5.8 ± 1.29

3. rGONR–PEG–cy3–RGD MTT assay 1–3 µm 1 µg ml−1 / U87MG 94% (light) [77]

NO
rGONR–PEG–cy3–RAD 24% (light)

17
NO

4. H/Cu/RGO MTT assay 2.20 ± 0.70 nm 7.8 µg ml−1 /24 h Normal CCD1 112 cells/cancer HCT11 cells 15%/50% [36]
15.6 µg ml−1 /24 h 5%/35%
31.2 µg ml−1 /24 h 0%/0%
62.5 µg ml−1 /24 h 0%/0%
125 µg ml−1 /24 h 0%/0%
250 µg ml−1 /24 h 0%/0%
500 µg ml−1 /24 h 0%/0%

5. rGO MTT assay — 25 µg ml−1 /24 h Cal33 & HCT-116 p53+/+ 75% 90% Present work
50 µg ml−1 /24 h 68% 74%
100 µg ml−1 /24 h 65% 72%
200 µg ml−1 /24 h 21% 38%
Cu/Glu/rGO synthesized at 100 ◦ C 25 µg ml−1 /24 h Cal33 & HCT-116 p53+/+ 62% 64%
50 µg ml−1 /24 h 50% 50%
100 µg ml−1 /24 h 14% 32%
200 µg ml−1 /24 h 3% 3%
Cu/Glu/rGO synthesized at 200 ◦ C 25 µg ml−1 /24 h Cal33 & HCT-116 p53+/+ 66% 66%
50 µg ml−1 /24 h 52% 55%
100 µg ml−1 /24 h 21% 36%
200 µg ml−1 /24 h 3% 4%
A Singh et al
Biomed. Mater. 18 (2023) 065025
Figure 8. Synthesis and mechanisms of antibacterial and anticancer activity for Cu/Glu/rGO nanocomposites.
5. Conclusion
The chemical reduction method using glucose
is effective in preparing glucose-capped copper
nanoparticle decorated reduced graphene oxide
(Cu/Glu/rGO) nanomaterial since this method
neither requires nor produces toxic substances, is
comparatively cost-effective, and can be performed
within a relatively short period (less than 2 h). In
general, copper nanoparticles possess good antibac-
terial activity but suffer from issues of high toxicity
and agglomeration. Here, the use of glucose and rGO
increases biocompatibility and controls the agglom-
eration of copper nanoparticles. Characterization of
nanomaterial via XRD, FTIR, TEM, and RAMAN
are significantly correlated with each other. Results
obtained in this work show that all the nanomater-
ial Cu/Glu, Cu/rGO, and Cu/Glu/rGO synthesized
at 100 ◦ C and 200 ◦ C have antibacterial and anti-
cancer properties. However, Cu/Glu/rGO is more
promising to be used as an antibacterial and antican-
cer agent assigned to the synergistic effect of copper,
glucose, and rGO. Also, glucose capping leads to the
formation of the Cu2 O form of copper that releases
Cu+ ions into the environment which can interact
with bacterial cells readily and kill them via different
mechanisms while the sharp corner basal planes of
rGO act as nano-knife and stimulate Cu2 O to gener-
ate more ROS leading to the exposure of intracellular
components followed by cell death.
Indeed, copper nanoparticles generate copper
ions in the environment which are utilized as a
cofactor for metalloproteins and enzymes, hence con-
tributing to the normal healthy processes of the
human tissues, yet, the use of metal ions for biomed-
ical applications is subjected to the removal of ions
from the body. Interestingly, Cu/Glu/rGO has shown
good cytotoxicity at low concentrations in both the
cancer cell lines tested, and it would be remarkable
to perform similar studies on the tumor. Results for
18
A Singh et al
Cu/Glu/rGO synthesized at 100 ◦ C show significant
antibacterial and anticancer activities at very low con-
centrations, suggesting this nanomaterial is probably
the best candidate for the management of bacterial
infections and cancer. Moreover, the local toxicity and
the long-term effects certainly need to be considered.
Data availability statement
All data supporting this study’s findings are included
within the article.
Acknowledgments
A S and A G are thankful to Advanced
Instrumentation Research Facility, Jawaharlal Nehru
University, and New Delhi, India for their X R D,
FT-IR T E M, and Raman facilities. A S and H R
K acknowledge the facilities and support provided
by the School of Biotechnology, Jawaharlal Nehru
University, New Delhi. A S and H R K acknow-
ledge by providing rGO by the School of Physical
Sciences, Jawaharlal Nehru University, New Delhi-
110067, India.
Funding
The work of AS was supported by the University
Grant Commission (UGC), grant number F.15-
1/2016-17/PDFWM-2015-17-UTT-33566 (SA-II).
This work was supported by the UGC BSR Research
Start-Up Grant, Grant Number F.30-473/2019 (BSR).
Conflict of interest
The authors have no relevant financial or non-
financial interests to disclose the work reports in this
paper.
Biomed. Mater. 18 (2023) 065025
ORCID iD
Hemant R Kushwaha  https://orcid.org/0000-
0002-0316-2799
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