Biotechnol Lett

DOI 10.1007/s10529-013-1239-x

REVIEW

Biogenic nanoparticles: copper, copper oxides, copper

sulphides, complex copper nanostructures and their
applications
Olga Rubilar • Mahendra Rai •
Gonzalo Tortella • Maria Cristina Diez •

Amedea B. Seabra • Nelson Durán

Received: 18 February 2013 / Accepted: 1 May 2013

Ó Springer Science+Business Media Dordrecht 2013

Abstract Copper nanoparticles have been the focus
of intensive study due to their potential applications in
diverse fields including biomedicine, electronics, and
optics. Copper-based nanostructured materials have
been used in conductive films, lubrification, nanofl-
uids, catalysis, and also as potent antimicrobial agent.
The biogenic synthesis of metallic nanostructured
nanoparticles is considered to be a green and eco-
friendly technology since neither harmful chemicals
nor high temperatures are involved in the process. The
present review discusses the synthesis of copper
nanostructured nanoparticles by bacteria, fungi, and
plant extracts, showing that biogenic synthesis is an
economically feasible, simple and non-polluting pro-
cess. Applications for biogenic copper nanoparticles
are also discussed.
Keywords Biogenic
Copper
Copper oxides

Copper sulphides
Nanoparticles
Introduction
Copper exhibits a broad-spectrum of biocidal activity
and effectively inhibits the growth of bacteria, fungi,
viruses and algae (Srivastava 2009; Grass et al. 2011;
Santo et al. 2012). During the last 2 years, nanoscale
copper has gained much attention due to its remark-
able antibacterial activity (Raffi et al. 2010; Cady et al.
2011; Chatterjee et al. 2012), and products with
copper-containing surfaces may be used for sterilisa-
tion processes in hospitals (Mikolay et al. 2010). In
contrast to silver nanoparticles that have been studied

O. Rubilar (&)
G. Tortella
M. C. Diez N. Durán

Department of Chemical Engineering, Temuco, Chile
and Scientifical & Technological Bioresource Nucleus,
Universidad de La Frontera, P.O. Box 54-D, Temuco,
Chile
e-mail: olga.rubilar@ufrontera.cl
M. Rai
Department of Biotechnology, Sant Gadge Baba Amravati
University, Amravati 444 602, Maharashtra, India
e-mail: mahendrarai7@gmail.com
Biological Chemistry Laboratory, Institute of Chemistry,
Universidade Estadual de Campinas, C. P. 6154,
Campinas, São Paulo 13083-970, Brazil
e-mail: duran@iqm.unicamp.br
N. Durán
Center of Natural and Human Sciences, Universidade
Federal do ABC, São Paulo, Brazil

A. B. Seabra
Exact and Earth Sciences Department, Universidade
Federal de São Paulo, Diadema, São Paulo, Brazil
e-mail: amedea.seabra@gmail.com

123

extensively for antibacterial application (Durán et al.
2010a, 2011; Prabhu and Poulose 2012), copper is an
essential element for living organisms and may be
suitable for biomedical applications. Moreover, cop-
per is cheaper (3.6 USD/lb) than silver (30 USD/lb)
and, therefore, a method utilising copper would prove
cost-effective (Jia et al. 2012).
The number one copper producer in the world,
Chile, which provides approximately one third of the
world’s copper, produced 5.24 million tons of the red
metal in 2011 (http://copperinvestingnews.com/
10581-chiles-copper-production-to-rise-in-2012.html).
Thus, Chile appears to have great potential for the
development of copper nanotechnology. An important
aspect of copper nanotechnology is the production of
nanostructures by eco-friendly and safe processes.
One of the processes that fulfils these requirements is
the biogenic synthesis of nanostructures.
There has been a surge of interest in the synthesis of
nanostructures using biological systems. Many
researchers have reported the biosynthesis of different
types of nanoparticles using diverse biological sys-
tems including bacteria, fungi, algae and plants
(Mandal et al. 2006; Mohanpuria et al. 2008; Rai
et al. 2008; Sinha et al. 2009; Durán et al. 2010a, b;
Narayanan and Sakthivel 2010, 2011; Blanco-Andujar
et al. 2010; Gade et al. 2010; Thakkar et al. 2010;
Durán et al. 2011; Marcato and Durán 2011; Li et al.
2011; Durán and Marcato 2012; Varshney et al. 2012;
Asmathunisha and Kathiresan 2013). In this context,
biogenic synthesis has made a significant contribution
to the production of metallic oxide nanoparticles
(Durán and Seabra 2012), metallic sulphides (Seabra
and Durán 2012), and nanostructures of iron deriva-
tives (Seabra et al. 2013). These processes represent an
important step toward the development of nanostruc-
tured materials.
This review discusses the biogenic synthesis of the
following copper derivative nanoparticles.
Cu0
Due to the toxicity of copper ions, biological systems
attempt to reduce the number of copper ions to prevent
copper toxicity. When grown in contaminated soil in
the natural environment, common plants can trans-
form copper into metallic nanoparticles in and near
their roots, with the assistance of endomycorrhizal
fungi (Manceau et al. 2008a, b). Due to their unique
123
Biotechnol Lett
physical and chemical properties and low cost of
preparation, copper nanoparticles have become of
great interest. Copper nanoparticles are currently used
in wound dressings and socks to give them biocidal
properties (Borkow et al. 2009, 2010). Furthermore,
copper nanoparticles have potential industrial appli-
cations, such as gas sensors, catalytic processes, high
temperature superconductors, solar cells and so on
(Li et al. 2008). Copper nanoparticles can be easily
oxidised to form copper oxide. If the application
requires copper nanoparticles to be protected from
oxidation, the copper nanoparticles are usually encap-
sulated in organic or inorganic material, such as
carbon, silica and multi-layered graphene (Athanas-
siou et al. 2006; Wang et al. 2012). In view of these
potentially interesting applications, copper nanoparti-
cles have been the focus of many investigations to
prepare them for commercial use (Singh et al. 2010;
Tran et al. 2010). Chemical synthesis with citrate can
modulate the synthesis of copper nanoparticles or
copper mixed with Cu2O particles, depending on the
reaction time (Bicer and Sisman 2010).
CuO, Cu2O, and Cu4O3
The synthesis strategy for CuO and Cu2O nanowire
fabrication starts with a precursor such as Cu(OH)2
nanowires. In particular, Cu2O and other related
materials are currently the subject of growing interest
for their potential use in high performance batteries.
Paramelaconite, Cu4O3, is a mineral that shows
puzzling magnetic properties. In a typical synthesis
process, copper foils are heated in the range of
200–1050 °C. In that range, it was noted that only one
phase, i.e., cuprous oxide, existed, while from 200 to
950 °C, a mixture of cuprous and cupric oxide was
observed (Matten 2008).
CuS
Variable stoichiometric compositions attract consid-
erable attention due to their many unusual electronic
and optic behaviours, demonstrating great potential
for use in a wide range of applications, including solar
cells, solar controllers, solar radiation absorbers,
catalysts, high-capacity cathode materials in lithium
secondary batteries, superconductors at low tempera-
ture, chemical sensors, and thermoelectric cool-
ing materials (Li et al. 2010; Iwahori et al. 2011).
Biotechnol Lett
These materials maintain transmittance in the infrared
region, and they have low reflectance in the visible
region and relatively high reflectance in the near
infrared region, making these nanoparticles suitable
for the development of optical filters, sensors, and
architectural glazes (Lu et al. 2011). Copper sulphide
nanoparticles have been produced by several methods
(Li et al. 2010); however, increasing interest in
developing an environment- friendly approach for
nanoparticle synthesis requires scientists to exploit
biological systems to manufacture nanoscale struc-
tures (Mukherjee et al. 2002; Basavaraja et al. 2008).
We now describe the importance of the biogenic
pathways of copper derivatives and their role in
technological and economically feasible processes.
Applications of copper-based nanostructured
materials
There has been an increasing awareness and interest in
biogenic synthesis of metallic nanoparticles because
of their multiple applications in various fields of
research, such as medicine, electronics, and optics
(Dewan et al. 2012; Gunalan et al. 2012). For example,
copper-based nanostructured materials can be used in
conductive films, lubrication, nanofluids, catalysis,
and also as potent microbicidal agents (Tilaki et al.
2007; Mitsudome et al. 2008; Longano et al. 2012).
The uses of copper-based nanomaterials are cost-
effective as compared to other metallic-based nanom-
aterials, such as gold or silver.
Copper-derived nanostructured materials have
remarkable catalytic efficiency (Mitsudome et al.
2008). Since copper is a low-cost metal with less
toxicity, and a copper-based catalyst can be recycled
and reused many times, the uses of copper nanopar-
ticles have been the focus of intense investigation in
the field of catalysis (Ramu 2012; Santhanalakshmi
and Parimala 2012; Svintsitskiy et al. 2013). The
efficacy of copper nanoparticles, dispersed in ionic
liquid comprised by ethylene glycol, as catalyst for the
synthesis of biologically active 3,4-dihydropyrimidi-
nones was reported (Dewan et al. 2012). The authors
further discussed the advantages and use of copper
nanoparticles as catalyst, since the reaction was
performed at room temperature, with reduced reaction
times, high yields, with no loss of nanoparticle
efficacy after multiple cycles of reaction. Huang
et al. (2012) reported the efficacy of zero-valent
copper nanoparticles employed as catalyst for the
dechlorination of dichloromethane, which is a recal-
citrant ground water contaminant. The degradation of
the contaminant was dependent on the dose of copper
nanoparticles. Figure 1 shows the kinetics of degra-
dation of dichloromethane by copper nanoparticles
and NaBH4. Dichloromethane was rapidly degraded
by copper nanoparticles in the presence of NaBH4,
indeed 90 % of dichloromethane was reduced within
1 h, while no degradation was observed in the
presence of either copper nanoparticles alone or
NaHB4 reduction conditions alone (Huang et al.
2012).
This result indicates the importance of copper
nanoparticles in environmental chemistry. In a similar
study, copper nanoparticles, synthesized from cupric
sulphate and hydrazine, showed potent catalytic action
on the reduction of a series of aromatic nitro
compounds to aromatic amino compounds (Duan
et al. 2012). Copper nanoparticles are efficient cata-
lysts with low-cost, high yields, easy product separa-
tion, and the nanoparticles can be reused many times
(Duan et al. 2012). Taken together, copper-based
nanomaterials have advantages in comparison with
traditional methods of catalysis.
In addition to the catalyst, copper-based nano-
structed materials are also important in electronic
devices, such as inkjet printing (Cheon et al. 2012):
well-dispersed and stable copper nanoparticles with an
average diameter of 15 nm were prepared by a
modified electrolysis method (Cheon et al. 2012).
Fig. 1 Degradation of dichloromethane (26.4 mg l-1) in the
presence of Cu0 nanoparticles (2.5 g l-1) and NaBH4 (1 g l-1).
(From Huang et al. 2012 by permission of Elsevier)
123

A water-based, copper nano-colloidal suspension was
prepared by using an amphiphilic surfactant and
printed onto a polyimide substrate using ink-jet
printer. The results revealed the writing of highly
conductive lines by using copper nanoparticles con-
ductive ink, which may find important applications in
the manufacturing of electronics, such as smart
packing, tags, and radio frequency identification
(Cheon et al. 2012). Similarly, well-dispersed and
uniform sized copper nanoparticles embedded in a
polyimide film were investigated for non-volatile
memory devices (Gupta et al. 2012). Copper nano-
particles embedded in polyimide film are relatively
inexpensive and have a very good performance,
highlighting the potential electronic application of
this material.
Another important property of nanostructed-based
copper material is its antimicrobial activity against a
wide-range of microorganisms, such as bacteria and
fungi (Ramyadevi et al. 2012). In this context, the
combination of copper bioactivity properties with
nanotechnology might result in a material with
desirable microbicidal properties for diverse applica-
tions, such as the protection of medical instruments,
water treatment and food processing (Veerapandian
et al. 2012). Longano et al. (2012) described the
fabrication of copper nanoparticles by laser, which
were further embedded in a biodegradable polymer
matrix comprised of polylactic acid, yielding a
nanocomposite. The nanocomposite thus obtained
possesses the antimicrobial properties of copper and
biocompatible properties of polylactic acid. The
antibacterial activity of this nanocomposite was eval-
uated against a mix of Pseudomonas spp., that
proliferate in processed food. The results indicate that
this copper-based nanocomposite can be used as an
additive for food packaging applications (Longano
et al. 2012). Similarly, Bajpai et al. (2012) reported the
preparation of copper nanoparticles loaded in alginate-
impregnated cotton fabric with bactericidal properties.
The fabrics released Cu2? in physiological fluids at
37 °C over 50 h. Moreover, the antibacterial activity
of fabric was evaluated against Escherichia coli by
modified agar diffusion assay (Bajpai et al. 2012).
An important biomedical application of copper
sulphide nanoparticles is as a new class of photoacou-
stic contrast agents for deep-tissue imaging (Ku et al.
2012). Intracranial injection of semiconducting copper
sulphide nanoparticles allowed visualization of mouse
123
Biotechnol Lett
brain in a photoacoustic tomography (Ku et al. 2012).
The use of this nanoparticle may therefore find
applications for molecular imaging of breast cancer.
A further interesting biomedical application of cop-
per-based nanostructure materials is as electrochem-
ical biosensors and bioelectronic devices (Srivastava
et al. 2013). Ultrafine, monodispersed cuprous oxide
nanoparticles capped with poly-N-vinylpyrrolidone
were synthesized and embedded into biocompatible
chitosan (Sing et al. 2013). This nanocomposite was
used to fabricate cholesterol biosensors through the
immobilization of cholesterol esterase and cholesterol
oxidase onto the ultrafine cuprous oxide-chitosan/
indium tin oxide electrode surface. This biosensor
could then measure total cholesterol level in human
serum (Singh et al. 2013). Finally, copper nanoparti-
cles/graphene oxide composites were employed for
surface-enhanced Raman scattering (SERS), with
potential applications for optical chemical sensor
(Zhang 2012). It must be noted that despite some
important investigations on the toxicity of copper-
based nanostructure materials (Gomes et al. 2013),
further exhaustive studies are still needed in order to
propose safe biomedical applications of this material.
Biogenic preparation of copper, copper oxides
and copper sulphide
Copper nanoparticles (Cu0)
Varshney et al. (2010) synthesised copper nanoparti-
cles using a biomass of Pseudomonas stutzeri in an
aqueous CuSO4 solution which were characterised by
high resolution transmission electronic microscopy
(HRTEM) and selected area electron diffraction:
(SAED). The copper nanoparticles were spherical,
with a thin, 8–15 nm, coating on all particles. This was
an indication that the bacterial surface acts both as a
reducing and capping agent. Moreover, these micro-
photographs also revealed that copper nanoparticles
were well dispersed and stable for months (Varshney
et al. 2010).
The biocatalytic reduction and removal of copper
ions (CuCl2) was observed in the aqueous/organic
phase after the induction of copper reductase in yeast
grown in catabolic repression conditions, following
the addition of an inducer (copper ions) during the
initial growth phase of Saccharomyces cerevisiae.
Biotechnol Lett
The excess of glucose produced under catabolic
repression conditions was used to induce Cyt P450
in the yeast. The harvested biomass was lysed using a
cell disrupter and microsomes were obtained. The
most important parameters for biocatalytic reduction
were the initial substrate concentration, time of
reaction, initial pH of the reaction mixture (optimisa-
tion of the enzyme), and the concentration and time of
addition of the inducer (yeast growth) (Chandran et al.
2001).
Silver-resistant bacteria, Morganella morganii RP4
and M. psychrotolerans, were grown in LB broth (pH
7) and, later, the biomass was treated with an aqueous
CuSO4 solution. The reaction was continued for an
additional 24 h. After the reaction, the bacteria were
removed by centrifugation, and the coloured superna-
tant containing the nanoparticles was analysed by
HRTEM, X-ray photoelectron spectroscopy (XPS),
Fourier transform infrared (FTIR), UV–Vis spectros-
copy and SAED. These analyses revealed that Mor-
ganella strains growing at pH 7 produced only copper
nanoparticles (3–10 nm) (Ramanathan et al. 2011).
Lee et al. (2011) reported the biosynthesis of copper
nanoparticles from leaf extracts of Magnolia in the
presence of CuSO4. UV–Vis spectroscopy was used to
monitor the quantitative formation of copper nano-
particles. The synthesised nanoparticles were charac-
terised with inductively coupled plasma (ICP),
energy-dispersive spectroscopy (EDS), XPS, trans-
mission electron microscopy (TEM) and scanning
electron microscopy (SEM) (40–100 nm).
Stable and uniform copper nanoparticles
(11–40 nm) were synthesised by the formation of a
complex Cu-soybean extract and reduction (pre-
formed copper oxide) by sodium borohydride. This
represents a simple process for obtaining copper
nanoparticles (Guajardo-Pacheco et al. 2010).
When grown in contaminated soil in the natural
environment, rhizospheres of Phragmites australis
and Iris pseudoacorus can transform copper into
metallic nanoparticles in and near roots, with assis-
tance from endomycorrhizal fungi (Manceau et al.
2008a, b). Biomolecular responses to oxidative stress,
similar to the reactions used to abiotically synthesise
copper nanostructures of a controlled size and shape,
can cause transformation. This newly identified mode
of copper biomineralisation by plant roots under
copper stress may be common in oxygenated envi-
ronments (Manceau et al. 2008a, b).
The synthesis of copper nanoparticles by the
P. stutzeri bacterial strain from electroplating waste-
water has been reported (Varshney et al. 2011). The
bacterial strain was isolated from soil and character-
ised by UV–Vis spectroscopy, X-ray diffraction
(XRD), SEM and energy-dispersive X-ray spectros-
copy (EDX), which revealed that it produced
50–150 nm sized cubical copper nanoparticles
(Varshney et al. 2011).
Copper nanoparticles with average diameter of
15 ± 1.7 nm were synthesised at room temperature
using an aqueous extract prepared from latex of
Calotropis procera L. that was challenged with a
copper acetate solution. These biosynthetic copper
nanoparticles showed excellent long-term stability in
an aqueous medium. The copper nanoparticles were
characterised by HRTEM, XRD patterns, energy
dispersive analysis of X-rays (EDAX) and FTIR
analyses (Harne et al. 2012). Figure 2 shows the
HRTEM microphotographs of copper nanoparticles
synthesized by (a) 0.1 %, (b) 0.3 % and (c) 0.5 %
aqueous extract prepared from C. procera L. latex.
Figure 2 (a, b) reveals that polydispersed spherical
particles were obtained with 0.1 and 0.3 % of aqueous
extract from C. procera L. latex, while 0.5 % aqueous
extract leads to the formation of spherical and
monodispersive nanoparticles (Fig. 2c) (Harne et al.
2012). These results indicate that the experimental
condition and parameters are able to control the
morphology of copper nanoparticles.
Microorganisms, such as Fusarium oxysporum
(93–115 nm) and Pseudomonas sp. (84–130 nm),
were able to leach copper from the integrated circuits
present on electronic boards under ambient conditions.
Lantana camara, a weed commonly found in Maha-
rashtra, was also screened for its ability to leach
copper. The characteristics of the copper nanoparticles
obtained were studied using XRD, EDS, SEM, FTIR,
TEM, thermogravimetric analysis and cyclic voltam-
metry (Majumber 2012).
Copper/copper oxides (CuO, Cu2O, and Cu4O3)
A bacterium, Serratia sp., was grown to the stationary
phase in LB broth medium, centrifuged, and the pellet
was resuspended in distilled water containing CuSO4.
The culture was incubated and the cells were removed
by centrifugation to collect the copper oxide nanopar-
ticles (Hasan et al. 2008). During this process,
123

Fig. 2 Transmission electron microphotographs of copper nanoparticles synthesized using a 0.1 %, b 0.3 %, and c 0.5 % aqueous
extract prepared from Calotropis procera L. latex. (From Harne et al. 2012 by permission of Elsevier)
the bacteria converted almost all of the salt into
nanoparticles, exhibiting a broad peak of the absorp-
tion spectra, probably due to the overlapping of the
absorption spectrum of copper oxide with that of
the residual CuSO4 (Hasan et al. 2008). In general, the
bacteria do not survive 5 h after the addition of the
copper salt. Normal bacterial survival was *15–20 h
in the absence of the salt. TEM images revealed that the
particles had a polydispersed distribution (10–30 nm
in diameter). SAED analysis showed a crystalline
nature of the nanoparticles, with spots corresponding
to the [111], [110], [112], and [313] planes of copper
oxide (Hasan et al. 2008). The XRD pattern
displayed intense Bragg’s reflections related to
metallic copper and different copper oxides, where
CuO was the predominant phase. EDX, XPS and
FTIR analyses corroborated this finding, and also
revealed the presence of carbon and nitrogen (pro-
tein). These results demonstrate that in the presence
of CuSO4, the bacterium, Serratia, was able to
123
Biotechnol Lett
produce a mixture of metallic copper and different
copper oxides (Hasan et al. 2008).
Copper oxide (Cu2O) nanoparticles were synthes-
ised using Lactobacillus sp. and S. cerevisiae. The
synthesis was performed at room temperature, and
individual nanoparticles were between 10 and 20 nm
(Prasad et al. 2010). A possible mechanism for the
synthesis of metallic oxide nanoparticles was also
proposed, where pH and the partial pressure of H2
(r-H2), or redox potential of the culture solution, seem
to play an important role in the process, as previously
reported for Sb2O3, by Jha et al. (2009).
Usha et al. (2010) reported a method for the green
synthesis of copper oxide by Streptomyces sp.
(100–150 nm). Isolation of this actinomycete was
performed by a soil-dilution plating technique, using
glycerol/yeast extract/agar.
Singh et al. (2010) reported the biological synthesis
of copper oxide nanoparticles using E. coli with
variable size and shapes. The precipitated protein
Biotechnol Lett
fraction of E. coli synthesised copper oxide nanopar-
ticles under simple experimental conditions, such as
an aerobic environment, neutral pH and room tem-
perature, that are important for the stabilisation of the
formed nanoparticles (Singh et al. 2010).
For the biogenic synthesis of copper oxides,
Penicillium aurantiogriseum, P. citrinum and
P. waksmanii were cultured in Czapek Dox broth
including sucrose and yeast extract and incubated at
200 rpm for 10 days at 28 °C. The cultures were
centrifuged and the supernatants were used to synthe-
sise copper nanoparticles. Thereafter, CuSO4 solution
was separately added to the supernatants of
P. aurantiogriseum, P. citrinum and P. waksmanii,
and incubated again for 24 h at 28 °C. The presence of
spherical copper oxide nanoparticles was determined
by SEM. Dynamic light scattering (DLS) showed that
at a pH range of 5–9, at the same copper salt
concentration; no large polydispersity was observed
(P. citrinum, diam. 91–133 nm; P. waksmanii, diam.
80–88 nm; and for P. aurantiogriseum, diam.
91–119 nm). These results were further supported by
UV–Vis and fluorescence spectrum analysis, which
revealed the presence of some proteins secreted by the
fungi in culture (Honary et al. 2012).
SEM images demonstrated that Cu2O (60–80 nm)
produced by Tridax procumbens leaf extract and
coated in the polymer, PANI, appeared to be hexag-
onal and cubic, with rough surfaces. The SEM image
of PANI coated Cu2O showed that the particles have a
core–shell structure, and their surface was coated with
a platelet-like polyaniline (Gopalakrisnan et al. 2012).
Sangeetha et al. (2012) produced mono-dispersed,
versatile and highly-stable CuO nanoparticles (size 15
and 30 nm) using an Aloe vera extract. SEM and TEM
image characterisation showed that there were no
changes in the shape of CuO nanoparticles produced
by A. vera extract compared with CuO nanoparticles
formed chemically. However, high concentration of A.
vera extract caused an increase in the CuO nanopar-
ticle size.
Cooper sulphides (CuS)
Spherical CuS nanoparticles were synthesised by
F. oxysporum (Hosseini et al. 2012) by treating the
fungus or its secretion with CuSO4 solution under mild
conditions. Nanoparticles (20 nm) were characterised
and the authors observed that the presence of proteins on
the nanoparticle surface stabilised the suspensions and
prevented particle aggregation (Hosseini et al. 2012).
Temperature affected chalcophile trace metal con-
taminants (Cu) upon flooding of a riparian soil that
was contaminated by past mining activities, and
colloidal metal sulphide formation dominated copper
pore water dynamics at high temperatures due to the
fast sulphate reduction. X-ray absorption fine structure
spectroscopy revealed metallic Cu0 as the main
colloidal copper species prior to sulphate reduction
at all temperatures. Analytical electron microscopy
showed that Cu0 particles were associated with the
suspended bacteria, suggesting the biomineralisation
of Cu0. Upon the onset of sulphate reduction, metallic
copper particles were transformed into CuxSy. These
results provided evidence that temperature controls
trace metal dynamics during soil flooding via its
influence on the microbial reduction of terminal
electron acceptors (Hofacker et al. 2012a, b).
Bioactive properties of copper derivatives
The antibacterial activity of Cu2O nanoparticles
(60–80 nm) synthesised from T. procumbens leaf
extract was evaluated against E. coli, as a model for
Gram-negative bacterium, by the disc-diffusion
method on LB agar plates containing different nano-
particle concentrations. At 20 lg/ml, bacterial growth
was inhibited by 65 %, while between 50 and 60 lg/
ml, bacterial growth was totally inhibited. The authors
suggested a possible mechanism for this inhibition
whereby Cu2O nanoparticles are absorbed on the cell
surface, impair the cell wall and then damage the cell
membrane, increasing the permeability of the cell
membrane and decreasing the viability of the bacteria
in the Cu2O solution (Gopalakrishnan et al. 2012).
Copper nanoparticles synthesised by using a Magnolia
leaf extract as a reducing agent (40–100 nm) showed
antibacterial activity against E. coli: foam coated with
biologically-synthesised copper nanoparticles showed
higher antibacterial activity compared with untreated
foam (Lee et al. 2011).
Copper nanoparticles have been synthesised using
aqueous extracts of C. procera L. latex (15 ± 1.7 nm).
The authors showed that anti-oxidant enzymes present
in the latex of C. procera L., cysteine protease with free
thiol (SH) group and tryptophan, played an important
role in reducing and stabilising copper nanoparticles.
123

The cytotoxicity of latex-stabilised copper nanoparti-
cles against HeLa, A549 and BHK21 cell lines was
determined using an MTT assay. These nanoparticles
showed excellent viability and biocompatibility up to
120 lM (Harne et al. 2012).
The copper oxide nanoparticles (100–150 nm) pro-
duced by Streptomyces sp. were used to develop
antimicrobial textiles that can be used in hospitals to
prevent or minimise infections from pathogenic bacte-
ria. The zone of mycostasis was used to determine the
antifungal activity of copper impregnated fabrics
against Aspergillus niger. In the control fabrics
(absence of copper nanoparticles), no effect was
observed but cotton fabrics with copper nanoparticles
displayed the maximum zone of mycostasis. The results
of the percentage reduction test corresponded with
those of the well diffusion method. A wash durability
test was also carried out with these test fabrics and
significant antimicrobial activity was retained in the
copper oxide, nanoparticle-treated fabrics for up to 8
washes. After eight washes, the percentage of antimi-
crobial activity was low, and no activity was observed
after 17 washes (Usha et al. 2010).
Copper nanoparticles from F. oxysporum
(93–115 nm), Pseudomonas sp. (84–130 nm) and
L. camara were effective against a hospital strain
E. coli 2065. Applications for eco-friendly nanoparticles
in antimicrobials, wound healing and other medical and
electronic applications make this method potentially
exciting for large-scale synthesis (Majumber 2012).
Biogenic complex copper derivatives
The synthesis of mixed metal nanoparticles by plants
suggests the possibility of using plants to synthesise
catalysts of specific compositions containing Au, Ag
and Cu as an alloy that are difficult to synthesise by
traditional methods. Metal-rich soil was produced by
applying solutions of gold chloride, silver nitrate and
copper chloride to sieved and dried agricultural soil.
The soil was dried and distributed into greenhouse
pots and it was watered to dampness daily for 2 weeks
to equilibrate the metals and the soil. Brassica juncea
seeds were then added to the pots. After 14 days, the
above-ground biomass was harvested, dried and
sieved to less than 180 lm. TEM, high-angle annular
dark-field (HAADF) and STEM-EDX, and these
123
Biotechnol Lett
methods combined with EDX analyses, indicated that
the nanoparticles in the plants consist of an alloy and
not a mixed phase of separate Au/Ag/Cu elements
(Haverkamp et al. 2007).
To prepare CuAlO2 using a fungus-based route,
Cu(NO3)2 and Al(NO3)3 were used as starting mate-
rials in the presence of an alkalotolerant and thermo-
philic fungus, Humicola sp. The harvested mycelial
mass was resuspended in an aqueous copper and
aluminum nitrate solution at pH 9. The CuAlO2
nanoparticles (5 nm) were characterised by XRD,
TEM, UV–Vis and FTIR analyses (Ahmad et al.
2007).
Final remarks
This review discusses the recent knowledge of nano-
particles produced from copper derivatives using
different biological systems, mainly bacteria, fungi,
plants extracts. Numerous reports of chemically and
physically synthesised copper and copper-based
nanomaterials exist that have not been included in
this review but, apparently until now, it has been
suggested that not enough data are available on the
biological synthesis of copper nanomaterials with size
and shape control. However, the present review shows
that this is not completely true because new strategies
exist that are simple, cost-effective, ecologically
friendly, scalable and are able to control the size and
shape of copper, based materials that have been
published in recent years.
Acknowledgements We would like to thank FAPESP, CNPq,
INOMAT (MCTI/CNPq), the Brazilian Network of Nano-
toxicology (MCTI/CNPq), NANOBIOSS (MCTI/CNPq) and
FONDECYT project 1130854 for their support.
References
Ahmad A, Jagadale T, Dhas V, Khan S, Patil S, Pasricha R, Ravi
V, Ogale S (2007) Fungus-based synthesis of chemically
difficult-to-synthesize multifunctional nanoparticles of
CuAlO2. Adv Mater 19:3295–3299
Asmathunisha N, Kathiresan K (2013) A review on biosynthesis
of nanoparticles by marine organisms. Colloids Surf B
103:283–287
Athanassiou EK, Grass RN, Stark WJ (2006) Large-scale pro-
duction of carbon-coated copper nanoparticles for sensor
applications. Nanotechnology 17:1668
Biotechnol Lett
Bajpai SK, Bajpai M, Sharma L (2012) Copper nanoparticles
loaded alginate-impregnated cotton fabric with antibacte-
rial properties. J Appl Polym Sci 126:E318–E325
Basavaraja S, Balaji SD, Lagashetty A, Rajasab AH, Venka-
taraman A (2008) Extracellular biosynthesis of silver
nanoparticles using the fungus Fusarium semitectum. Mat
Res Bull 43:1164–1170
Bicer M, Sisman I (2010) Controlled synthesis of copper nano/
microstructures using ascorbic acid in aqueous CTAB
solution. Powder Technol 198:279–284
Blanco-Andujar C, Tung LD, Thanh NTK (2010) Synthesis of
nanoparticles for biomedical applications. Annu Rep Prog
Chem Sect A 106:553–568
Borkow G, Zatcoff RC, Gabbay J (2009) Reducing the risk of
skin pathologies in diabetics by using copper impregnated
socks. Med Hypotheses 73:883–886
Borkow G, Gabbay J, Dardik R, Eidelman AI, Lavie Y, Grun-
feld Y, Ikher S, Huszar M, Zatcoff RC, Marikovsky M
(2010) Molecular mechanisms of enhanced wound healing
by copper oxide-impregnated dressings. Wound Repair
Regen 18:266–275
Cady NC, Behnke JL, Strickland AD (2011) Copper-based
nanostructured coatings on natural cellulose: nanocom-
posites exhibiting rapid and efficient inhibition of a multi-
drug resistant wound pathogen, A. baumannii, and mam-
malian cell biocompatibility in vitro. Adv Funct Mater
21:2506–2514
Chandran CB, Subramanian TV, Felse PA (2001) Chemometric
optimization of parameters for biocatalytic reduction of
copper ion by a crude enzyme lyzate of Saccharomyces
cerevisiae grown under catabolic repression conditions.
Biochem Eng J 8:31–37
Chatterjee AK, Sarkar RK, Chattopadhyay AP, Aich P, Cha-
kraborty R, Basu T (2012) A simple robust method for
synthesis of metallic copper nanoparticles of high anti-
bacterial potency against E. coli. Nanotechnology
23:85–103
Cheon J, Lee J, Kim J (2012) Inkjet printing using copper
nanoparticles synthesized by electrolysis. Thin Solid Films
520:2639–2643
Dewan M, Kumar A, Saxena A, De A, Mozumdar S (2012)
Biginelli reaction catalyzed by copper nanoparticles. PLoS
ONE 7:e43078
Duan Z, Ma G, Zhang W (2012) Preparation of copper nano-
particles and catalytic properties for the reduction of aro-
matic nitro compounds. Bull Korean Chem Soc 33:
4003–4006
Durán N, Marcato PD (2012) Biotechnological routes to
metallic nanoparticles production: mechanistics aspects,
antimicrobial activity, toxicity and industrial applications.
In: Rai M, Cioffi N (eds) Nano-antimicrobials: progress
and prospects, vol Part 3. Springer, Berlin, pp 337–374
Durán N, Seabra AB (2012) Metallic oxide nanoparticles: state
of the art in biogenic syntheses and their mechanisms. Appl
Microbiol Biotechnol 95:275–288
Durán N, Marcato PD, De Conti R, Alves OL, Costa FTM,
Brocchi M (2010a) Potential use of silver nanoparticles on
pathogenic bacteria, their toxicity and possible mecha-
nisms of action. J Braz Chem Soc 21:949–959
Durán N, Marcato PD, Ingle A, Gade A, Rai M (2010b) Fungi
mediated synthesis of silver nanoparticles: characterization
processes and applications. In: Rai M, Kövics G (eds)
Progress in Mycology. Scientific Publishers, Jodhpur,
pp 425–449
Durán N, Marcato PD, Durán M, Yadav A, Gade A, Rai M (2011)
Mechanistic aspects in the biogenic synthesis of extracel-
lular metal nanoparticles by peptides, bacteria, fungi and
plants. Appl Microbiol Biotechnol 90:1609–1624
Gade A, Ingle A, Whiteley C, Rai M (2010) Mycogenic metal
nanoparticles: progress and applications. Biotechnol Lett
32:593–600
Gomes T, Araújo O, Pereira R, Almeida AC, Cravo A, Bebianno
MJ (2013) Genotoxicity of copper oxide and silver nano-
particles in the mussel Mytilus galloprovincialis. Mar
Environ Res 84:51–59
Gopalakrishnan K, Ramesh C, Ragunathan V, Thamilselvan M
(2012) Antibacterial activity of Cu2O nanoparticles on
E. coli synthesized from Tridax procumbens leaf extract
and surface coating with polyaniline. Digest J Nanomat
Biostruct 7:833–839
Grass G, Rensing C, Solioz M (2011) Metallic copper as an
antimicrobial surface. Appl Environ Microbiol 77(5):
1541–1547
Guajardo-Pacheco Ma J, Morales-Sanchez JE, Gonzalez-Her-
nandez J, Ruiz F (2010) Synthesis of copper nanoparticles
using soybeans as a chelant agent. Mat Lett 64:1361–1364
Gunalan S, Sivaraj R, Venckatesh R (2012) Aloe barbadensis
Miller mediated green synthesis of mono-disperse copper
oxide nanoparticles: optical properties. Spectrochim Acta
A 97:1140–1144
Gupta RK, Kusuma DYK, Lee OS, Srinivasan MP (2012)
Copper nanoparticles embedded in a polyimide film for
non-volatile memory applications. Mater Lett 68:287–289
Harne S, Sharma A, Dhaygude M, Joglekar S, Kodam K,
Hudlikar M (2012) Novel route for rapid biosynthesis of
copper nanoparticles using aqueous extract of Calotropis
procera L. latex and their cytotoxicity on tumor cells.
Colloids Surf B 95:284–288
Hasan SS, Singh S, Parikh RY, Dharne MS, Patole MS, Prasad
BLV, Shouche YS (2008) Bacterial synthesis of copper/
copper oxide nanoparticles. J Nanosci Nanotechnol 8:
3191–3196
Haverkamp RG, Marshall AT, van Agterveld D (2007) Pick
your carats: nanoparticles of gold–silver–copper alloy
produced in vivo. J Nanopart Res 9:697–700
Hofacker AF, Voegelin A, Kaegi R, Weber F-A, Kretzschmar R
(2012a) Temperature-dependent formation of metallic
copper and metal sulfide nanoparticles during flooding of a
contaminated soil. Geochim Cosmochim Acta. (Available
online 15 November 2012)
Hofacker A, Voegelin A, Behrens S, Kappler A, Kaegi R,
Kretzschmar R (2012b) Biogenic copper and metal sul-
phide colloid formation in a contaminated floodplain soil.
14.5 Abstract Volume: 10th Swiss Geoscience Meeting.
Environmental Biogeosciences, Bern, 16–17th November
2012; Abstr. 14.5
Honary S, Barabadi H, Gharaeifathabad E, Naghibi F (2012)
Green synthesis of copper oxide nanoparticles using Pen-
icillium aurantiogriseum, Penicillium citrinum and Peni-
cillium waksmanii. Dig J Nanomat Biostruct 7:999–1005
Hosseini MR, Schaffie M, Pazouki M, Darezereshki E, Ranjbar
M (2012) Biologically synthesizeised copper sulfide
123

nanoparticles: production and characterization. Mat Sci
Semicond Proc 15:222–225
Huang C-C, Lo S-L, Lien H-L (2012) Zero-valent copper
nanoparticles for effective dechlorination of dichloro-
methane using sodium borohydride as a reductant. Chem
Eng J 203:95–100
Iwahori K, Takagi R, Kishimoto N, Yamashita I (2011) A size
controlled synthesis of CuS nano-particles in the protein
cage, apoferritin. Mat Lett 65:3245–3247
Jha AK, Prasad K, Prasad K (2009) Biosynthesis of Sb2O3
nanoparticles: a low-cost green approach. Biotechnol J
4:1582–1585
Jia B, Mei Y, Cheng L, Zhou J, Zhang L (2012) Preparation of
copper nanoparticles coated cellulose films with antibac-
terial properties through one-step reduction. Appl Mat
Iterfaces 4:2897–2902
Ku G, Zhou M, Song SL, Huang Q, Hazle J, Li C (2012) Copper
sulfide nanoparticles as a new class of photoacoustic con-
trast agent for deep tissue imaging at 1064 nm. ACS Nano
6:7489–7496
Lee HJ, Lee G, Jang NR, Yun JM, Song JY, Kim BS (2011)
Biological synthesis of copper nanoparticles using plant
extract. Nanotech-2011 (NSTI Publ.). 1:371–374
Li L, Liang J, Tao Z, Chen J (2008) CuO particles and plates:
synthesis and gas-sensor application. Mater Res Bull
43:2380–2385
Li F, Wu J, Qin Q, Li Z, Huang X (2010) Controllable synthesis,
optical and photocatalytic properties of CuS nanomaterials
with hierarchical structures. Powder Technol 198:267–274
Li YJ, Chiu CY, Huang Y (2011) Biomimetic synthesis of
inorganic materials and their applications. Pure Appl Chem
83:111–125
Longano D, Ditaranto N, Cioffi N, Di Niso F, Sibillano T,
Ancona A, Conte A, Del Nobile MA, Sabbatini L, Torsi L
(2012) Analytical characterization of laser-generated
copper nanoparticles for antibacterial composite food
packaging. Anal Bioanal Chem 403:1179–1186
Lu Y, Meng X, Yi G, Jia J (2011) In situ growth of CuS thin
films on functionalized self-assembled monolayers using
chemical bath deposition. J Colloid Interface Sci 356:
726–733
Majumber DR (2012) Bioremediation: copper Nanoparticles from
electronic-waste. Inter J Eng Sci Technol 4:4380–4389
Manceau A, Nagy KL, Marcus MA, Lanson M, Geoffroy N,
Jacquet T, Kirpichtchikova T (2008a) Formation of
metallic copper nanoparticles at the soil-root interface.
Environ Sci Technol 42:1766–1777
Manceau A, Nagy KL, Marcus MA, Lanson M, Geoffroy N,
Jacquet T, Kirpichtchikova T (2008b) Formation of
metallic copper nanoparticles at the soil-root interface.
Environ Sci Technol 42(5):1766–1772
Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Muk-
herjee P (2006) The use of microorganisms for the for-
mation of metal nanoparticles and their application. Appl
Microbiol Biotechnol 69:485–492
Marcato PD, Durán N (2011) Biogenic silver nanoparticles:
applications in medicines and textiles and their health
implications. In: Rai M, Durán N (eds) Metal nanoparticles
in microbiology. Springer, Germany, pp 249–267
Matten A (2008) Some methodologies used for the synthesis of
cuprous oxide: a review. J Pak Mater Soc 2:40–43
123
Biotechnol Lett
Mikolay A, Huggett S, Tikana L, Grass G, Braun J, Nies DH
(2010) Survival of bacteria on metallic copper surfaces in a
hospital trial. Appl Microbiol Biotech 87:1875–1879
Mitsudome T, Mikami Y, Ebata K, Mizugaki T, Jitsukawa K,
Kaneda K (2008) Copper nanoparticles on hydrotalcite as a
heterogeneous catalyst for oxidant-free dehydrogenation of
alcohols. Chem Commun 39:4804–4806
Mohanpuria P, Rana NK, Yadav SK (2008) Biosynthesis of
nanoparticles: technological concepts and future applica-
tions. J Nanopart Res 10:507–517
Mukherjee P, Senapati S, Mandal D, Ahmad A, Islamkhan M,
Kumar R, Sastry M (2002) Extracellular synthesis of gold
nanoparticles by the fungus Fusarium oxysporum. Chem
Bio Chem 3:461–463
Narayanan KB, Sakthivel N (2010) Biological synthesis of
metal nanoparticles by microbes. Adv Colloid Interface Sci
156:1–13
Narayanan KB, Sakthivel N (2011) Green synthesis of biogenic
metal nanoparticles by terrestrial and aquatic phototrophic
and heterotrophic eukaryotes and biocompatible agents.
Adv Colloid Interface Sci 169:59–79
Prabhu S, Poulose E (2012) Silver nanoparticles: mechanism of
antimicrobial action, synthesis, medical applications, and
toxicity effects. Intern Nano Lett 2:32
Prasad K, Jha AK, Prasad K, Kulkarni AR (2010) Can microbes
mediate nano-transformation? Indian J Phys 84:1355–1360
Raffi M, Mehrwan S, Bhatti TM, Akhter JI, Hameed A, Yawar
W, Hasan MM (2010) Investigations into the antibacterial
behavior of copper nanoparticles against Escherichia coli.
Ann Microbiol 60:75–80
Rai M, Yadav A, Gade A (2008) Current trends in phytosyn-
thesis of metal nanoparticles. Crit Rev Biotechnol 28:
277–284
Ramanathan R, Bhargava SK, Bansal V (2011) Biological
synthesis of copper/copper oxide nanoparticles. Chemca
Conference 466. www.conference.net.au/chemeca2011/
papers/466.pdf
Ramu VG, Bordoloi A, Nagaiah TC, Schuhmann W, Muhler M,
Cabrele C (2012) Appl Catal A 431–432:88–94
Ramyadevi J, Jeyasubramanian K, Marilani A, Rajakumar G,
Rahuman AA (2012) Mat Lett 71:114–116
Sangeetha G, Rajeshwari S, Rajendran V (2012) Aloe barbad-
ensis Miller mediated green synthesis of mono-disperse
copper oxide nanoparticles: optical properties. Spectroc-
hima Acta Part A 97:1140–1144
Santhanalakshmi J, Parimala L (2012) The copper nanoparticles
catalysed reduction of substituted nitrobenzenes: effect of
nanoparticle stabilizers. J Nanopart Res 14:1090
Santo CE, Quaranta D, Grass G (2012) Antimicrobial metallic
copper surfaces kill Staphylococcus haemolyticus via
membrane damage. Microbiologyopen 1(1):46–52
Seabra AB, Durán N (2012) Microbial syntheses of metallic sul-
fide nanoparticles: an overview. Curr Biotechnol 1:287–296
Seabra AB, Haddad P, Durán N (2013) Biogenic synthesis of
nanostructurated iron compounds: Applications and per-
spectives. IET-Nanobiotechnology in press
Sing J, Srivastava M, Roychoudhury A, Lee DW, Lee SH, Mal-
hotra BD (2013) Bienzyme-functionalized monodispersed
biocompatible cuprous oxide/chitosan nanocomposite plat-
form for biomedical application. J Phys Chem B 117:
141–152
Biotechnol Lett
Singh AV, Patil R, Anand A, Milani P, Gade WN (2010) Bio-
logical synthesis of copper oxide nano particles using
Escherichia coli. Curr Nanosci 6:365–369
Sinha S, Pan L, Chanda P, Sen SK (2009) Nanoparticles fabri-
cation using ambient biological resources. J Appl Biosci
19:1113–1130
Srivastava A (2009) Antiviral activity of copper complexes of
isoniazid against RNA tumor viruses. Resonance 14(8):
754–760
Srivastava M, Sing J, Mishra RK, Ojha AK (2013) Electro-
optical and magnetic properties of monodispersed colloidal
Cu2O nanoparticles. J Alloy Compd 555:123–130
Svintsitskiy DA, Chupakhin AP, Slavinskaya EM, Stonkus
OA, Stadnichenko AI, Koscheev SV, Boronin AI (2013)
Study of cupric oxide nanopowders as efficient catalysts
for low-temperature CO oxidation. J Mol Catal A
368–369:95–106
Thakkar KN, Mhatre SS, Parikh RY (2010) Biological synthesis
of metallic nanoparticles. Nanomedicine NBM 6:257–262
Tilaki RM, Iraji Zad A, Mahadavi SM (2007) Size, composition
and optical properties of copper nanoparticles prepared by
laser ablation in liquids. Appl Phys A 88:415–419
Tran N, Mir A, Mallik D, Sinha A, Nayar S, Webster TJ (2010)
Bacterial effect of iron oxide nanoparticles on Staphylo-
coccus aureus. Int J Nanomed 5:277–283
Usha R, Prabu E, Palaniswamy M, Venil CK, Rajendran KR
(2010) Synthesis of metal oxide nano particles by Strep-
tomyces sp. for development of antimicrobial textiles. Glob
J Biotechnol Biochem 5:153–160
Varshney R, Bhadauria S, Gaur MS, Pasricha R (2010) Char-
acterization of copper nanoparticles synthesized by a novel
microbiological method. JOM-J Miner Met Mater Soc
62:102–104
Varshney R, Bhadauria S, Gaur MS, Pasricha R (2011) Copper
nanoparticles synthesis from electroplating industry efflu-
ent. Nano Biomed Eng 3:115–119
Varshney R, Bhadauria S, Gaur MS (2012) A review: biological
synthesis of silver and copper nanoparticles. Nano Biomed
Eng 4:99–106
Veerapandian M, Sadhasivam S, Choi J, Yun K (2012) Gluco-
samine functionalized copper nanoparticles: preparation,
characterization and enhancement of anti-bacterial activity
by ultraviolet irradiation. Chem Eng J 209:558–567
Wang S, Huang X, He Y, Huang H, Wu Y, Hou L, Liu X, Yang
T, Zou J, Huang B (2012) Synthesis, growth mechanism
and thermal stability of copper nanoparticles encapsulated
by multi-layer graphene. Carbon 50:2119–2125
Zhang K (2012) Fabrication of copper nanoparticles/graphene
oxide composites for surface-enhanced Raman scattering.
Appl Surf Sci 258:7327–7329
123