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Rapid synthesis of highly stable silver

nanoparticles and its application for
colourimetric sensing of cysteine
a a a
Sainath Babu , Michelle O. Claville & Kesete Ghebreyessus
a
Department of Chemistry, Hampton University, Hampton, VA
23668, USA
Published online: 03 Jan 2015.

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To cite this article: Sainath Babu, Michelle O. Claville & Kesete Ghebreyessus (2015): Rapid
synthesis of highly stable silver nanoparticles and its application for colourimetric sensing of
cysteine, Journal of Experimental Nanoscience, DOI: 10.1080/17458080.2014.994680

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 Journal of Experimental Nanoscience, 2014
http://dx.doi.org/10.1080/17458080.2014.994680

Rapid synthesis of highly stable silver nanoparticles and its application

for colourimetric sensing of cysteine
Sainath Babu#, Michelle O. Claville and Kesete Ghebreyessus*,#

Department of Chemistry, Hampton University, Hampton, VA 23668, USA

(Received 13 August 2014; final version received 26 November 2014)

A modified green approach for the synthesis of stable silver nanoparticles

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(AgNPs) using tea leaf extract is described. The method involves the reduction of
silver salt by the polyphenols present in the green tea leaf extract and requires no
additional capping/stabilising agents. Compared to other biogenic methods for
the synthesis of AgNPs, the uniqueness of the approach described here lies in its
simplicity, low-cost, and rapid synthesis rate; the reaction being completed within
10 15 min at room temperature. The reaction was carried out in alkaline medium
without stirring and heating, and requires no special cleaning or drying of the
glassware used. The synthesised AgNPs were characterised by UV Vis
spectroscopy and transmission electron microscopy (TEM). The results showed
that AgNPs with a strong surface plasmon resonance peak around 410 nm and
particle size in the 5 30 nm range were prepared. The synthesised AgNPs show
excellent chemical stability for more than six months in aqueous solution.
Additionally, we showed that the as-synthesised AgNPs can be used as highly
selective colorimetric and optical sensors for the detection of cysteine. Thus, with
a simple synthesis strategy, and enhanced stability, these green-tea-functionalised
AgNPs have the potential for further applications as biosensors and
antimicrobial agents.
Keywords: colorimetric sensors; metal nanoparticles; naked eye; amino acids;
green methods

1. Introduction
Noble metal nanoparticles of gold (AuNPs) and silver (AgNPS) have attracted
considerable attention because of their characteristic surface plasmon resonance that
occurs in the visible region, and their potential applications in several areas ranging
from colorimetric sensors, catalysis, bioimaging, environmental remediation, and cancer
therapy.[1 13] Among the nanomaterials, AgNPs are the most highly commercialised and
extensively studied nanoparticles due to their unique antibacterial and optoelectronic
properties. They are widely used in numerous consumer products, such as medical devices,
cleaning agents, personal care products, and textiles.[14 17] The conventional method for
the synthesis of AgNPs involves the reduction of silver ions by reducing agents such as
sodium citrate or sodium borohydride, followed by the addition of capping agents such as
alkanethiols and alkylamines, or polymeric materials such as polyvinyl alcohol, and

*Corresponding author. Email: kesete.ghebreyessus@hamptonu.edu

#
Both authors contributed equally to this work.

Ó 2014 Taylor & Francis

 2 S. Babu et al.

gelatin to stabilise the AgNPs from aggregation.[17 20] However, the use of capping
agents for stabilisation and toxic chemicals for reductions may result in the formation of
hazardous by-products, adversely affecting their use in the biomedical fields and chemical
applications.
In order to overcome these drawbacks, the application of green methods to the
synthesis of nanoparticles is rapidly expanding as the broader utility of such approaches is
realised.[10,11,14,15,17,21 33] A recent review by Varma et al. [21] has highlighted the
applicability of green synthetic approaches using plant extracts, enzymes, micro-
organisms, biodegradable polymers, and microwaves to prepare AgNPs through redox
reactions. While many themes and variations have been developed, the use of plant-based
extracts as both reducing and capping agents has emerged as a simple and attractive
method for the synthesis of AgNPs. This new technique for the green synthesis of AgNPs
offers the distinct advantages of being environmentally friendly, low- cost, and able to
generate nanoparticles with good biocompatibility. To date, many plant parts including
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leaves, seeds, stems, roots, and fruits have been successfully used for the synthesis of
nanoparticles.[17,21 35] However, in most cases, longer reaction times (2 5 h) and
constant stirring and heating requirements present some major drawbacks in those
approaches.[12,25,26] To our knowledge, the only successful method that describes a fast
synthesis rate of AgNPs, with a reaction time of 15 min or less, was recently reported by
Wang et al.,[32] and involves the use of pear fruit juice as a reducing and capping agent.
Hence, there is a sustained need for the development of simple and rapid synthetic
methods for nanoparticles using eco-friendly natural products based on the principles of
green chemistry.
Herein, we report the facile green synthesis of AgNPs using green tea leaf extracts as
reducing and stabilising agents under alkaline conditions. Compared with the previously
reported approaches, the method described here is straightforward, simple, reproducible,
cost effective, and rapid. The synthesis was completed within 10 15 min, producing
brown-coloured colloidal solutions of AgNPs at room temperature. The synthesised
AgNPs are highly stable, staying without any noticeable change for more than six months
while kept at room temperature and without any protection from light exposure. We have
also explored the potential applications of the synthesised AgNPs as colourimetric and
optical probes for the detection of cysteine in aqueous solution.
Cysteine was chosen as a detecting target, because sulphur-containing amino acids such
as cysteine and homocysteine are essential biomolecules required for the normal growth of
cells and tissues in living systems.[36 38] These biological thiols play a significant role in
living systems by providing important insights into the proper physiological functions and
diagnosis of diseases. For instance, a variance in normal levels of cysteine is implicated
with several health problems such as slowed growth, hair depigmentation, oedema,
lethargy, liver damage, muscle and fat loss, skin lesions, and weakness.[39,40] Abnormal
levels of homocysteine in human plasma have been implicated in incidences of
cardiovascular and Alzheimer’s diseases.[39,40] Therefore, it is of great interest to develop
a selective and sensitive method for the detection of thiol-containing amino acids in
biological samples.
Typical methods for the detection of sulphur-containing amino acids include
high-performance liquid chromatography,[37,40,41] mass spectrometry,[42] capillary
electrophoresis,[41] electrochemical methods,[39,43] and some colourimetric assays.[5 9]
Most of the reported detection methods need complicated and expensive instruments and
 Journal of Experimental Nanoscience 3

require time-consuming pretreatment procedures for analysis. Compared to the

instrumental analysis, the colourimetric detection method is more convenient and efficient
for thiol detecting.[5 9,44] In recent years, some colourimetric assays based on cysteine-
induced aggregation of AgNPs have been reported as nanosensor probes for the detection
of cysteine.[5 9,32] However, most of the above detection processes rely on the addition
of polymeric surfactants as capping agents or metal ions to enhance the sensitivity and
selectivity of the AgNP probes.[5 9,32] In the present study, highly stable green-tea-
capped AgNPs have been synthesised and used as colourimetric and optical probe for the
selective detection of cysteine. The absorbance of the AgNPs was quenched efficiently in
the presence of cysteine. The assay for cysteine was not only sensitive and selective but
also simple, cost effective, and environmentally friendly.
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2. Materials and methods

2.1. Materials
All the chemicals and reagents used in this study were purchased from commercial sources
and used as received. Green tea (Lipton, USA) was purchased from a local store. High-
purity water (Milli-Q 18.2 MV cm) was used for preparing the AgNPs and stock solutions
throughout this study. All the amino acid solutions and reagents were freshly prepared
and used on the same day.

2.2. Instruments
UV Vis absorption spectra of the AgNPs were recorded on a Varian Cary 50 BIO
spectrometer. Transmission electron microscopy (TEM) images were recorded on a JEOL
high-resolution transmission electron microscope (HRTEM-JEM-2100F) instrument at
200 kV accelerating voltage. The microscope was equipped with a charge-coupled device (CCD)
digital camera, and a carbon-coated copper grid was used for sample preparation. The
configuration and crystallinity of the AgNPs were determined by X-ray diffraction (XRD)
(Rigaku, MiniFlex II), operated at a voltage of 40 kV and a current of 30 mA with Cu Ka
radiation. All the pictures of the AgNPs and cysteine mixtures were taken by Samsung
Galaxy SIII camera (Samsung, USA).

2.3. Preparation of the tea extract

Dry green tea leaves (400 mg) were added to 30 mL water in 150-mL Erlenmeyer flask. The
mixture was vigorously stirred over a magnetic stir plate for 30 min at room temperature
and filtered with Whatman 1 filter paper. The freshly prepared tea extract was then used
for the synthesis of AgNPs as described below.

2.4. Synthesis of AgNPs using green tea extract

Well-defined silver nanoparticles were synthesised using freshly prepared aqueous
solutions of green tea leaf extract in the following manner.[43] The stock solution of the
tea leaf extract was diluted to 5%, 10%, 20%, 30%, and 40% (V/V) with deionised water.
 4 S. Babu et al.

Next, 1.00 mL of silver nitrate (1 mM) and 0.25 mL of NaOH (0.10 M) solutions were
added dropwise in sequence into the dilute tea extract solutions. The contents of the
reaction vessels were combined with gentle swirling, and brown AgNPs were produced
immediately at room temperature under ambient light conditions.

2.5. Detections of amino acid with AgNPs

A solution of 80 mL of the green-tea-capped AgNPs was diluted to 3 mL of the total
volume in a quartz cuvette. To this solution, 5 mL (1 mM) cysteine was added, and the
contents of the solution were mixed by using a disposable pipette. The mixture was kept
3 5 min at room temperature for equilibration prior to UV Vis analysis. The UV Vis
absorption spectra of the samples were scanned from 230 to 700 nm. The same procedure
was followed for studying the interaction of the various amino acids and biomolecules
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with AgNPs. In addition, the interaction of cysteine with the AgNPs in the presence of
70 mM NaCl was studied following the same procedure.

3. Results and discussion

3.1. Synthesis and characterisation of the AgNPs
Colloidal AgNPs have been synthesised using an aqueous solution of green tea leaf extract
as a reducing and capping agent at room temperature and alkaline conditions, in an eco-
friendly manner. The reaction for the synthesis of the AgNPs was carried out in alkaline
media, because stable AgNPs could not be produced under acidic or neutral solutions
(data no shown). It is known that green tea contains various polyphenolic compounds and
phytochemicals, the prime constituents being theaflavins.[45] These polyphenolic
compounds can be easily deprotonated under alkaline conditions and serve as both
reducing and stabilising agents.[5,32,46] In this regard, the NaOH solution was added to
provide high alkalinity and enhance the oxidation capacity of AgNO3. The formation of
AgNPs was indicated by the immediate colour change from light yellow to dark brown
upon the addition of 0.10 M NaOH. The effect of the amount of tea leaf extract on the
formation of AgNPs was also studied. The amount of silver nitrate was kept constant
(1.0 mL, 1 mM), and the amount of tea extract was varied as 5%, 10%, 20%, 30%, and
40% (V/V). The intensity of the brown colour increased with the increase in the
concentration of the tea extract (Figure 1(b)).
As described earlier, longer reaction time is one of the major drawbacks for most of the
plant-extract-based synthetic methods. In most cases, the reaction ranges from 2 to 5 h
and involves continuous stirring and heating. In contrast, this study demonstrates rapid
synthesis of the AgNPs completed within 10 15 min, and confirmed by the instantaneous
colour change from light yellow to brown. The colour change indicates the reduction of
AgC ions to AgNPs under alkaline conditions. Furthermore, this green synthesis of
AgNPs proceeds without the need for any external heating or stirring. Thus, this technique
proves to be simple, inexpensive, and efficient, since no heat or any additional surfactants
or polymers as stabilising agents are needed.
The formation of the tea-extract-stabilised AgNPs was also confirmed by UV Vis
spectroscopy. The prepared AgNPs exhibited a characteristic surface plasmon resonance
band centred around 410 nm, an absorption wavelength that is insensitive to the
 Journal of Experimental Nanoscience 5
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Figure 1. (Colour online) (a) UV Vis absorption spectra of colloidal solution of AgNPs with
different concentrations of green tea leaf extract (5% 40%). (b) Visual images of the AgNPs with
different concentrations of tea leaf extracts at room temperature.

concentration of tea leaf extract. As shown in Figure 1(b), although different

concentrations of tea extract gave different colours, no shift was observed in the λmax
values of 5% 40% tea leaf extracts (Figure 1(a)). This indicates that nearly the same sizes
of AgNPs are formed regardless of the concentration of the tea extract. However, the
number of AgNPs formed at a high concentration of tea extract is higher than that of a
lower concentration, as evidenced by higher absorbance values. Reported examples
indicate that the increase in absorbance values with the increase in the concentration of tea
extract demonstrates higher production of AgNPs.[10,47] The symmetry of the plasmon
resonance absorption bands as shown in Figure 1(a) also indicates the formation of mostly
uniform AgNPs with low levels of agglomeration in solution.[48]
The formation of the AgNPs was further confirmed by TEM. Figure 2 shows TEM
images of AgNPs synthesised using 20% green tea leaf extract (V/V%). The as-synthesised
particles are predominately spherical in shape with very few having irregular shapes of flat
crystals (Figure 2). The size of the particles was 5 30 nm.
The crystalline nature of the AgNPs was also confirmed by XRD. The XRD patterns
of the dried samples of the AgNPs are shown in Figure 3. The diffraction peaks at
2u degrees of 38.02, 44.12, 64.36, and 77.36 can be attributed to the reflections of (111),
(200), (220), and (311) planes of the face-centred cubic crystalline structure of metallic
silver, respectively. In addition, the minor peak near 32.10 implied the possible existence
of Ag2O in the sample.[17,49]

3.2. The stability of the AgNPs

The synthesised AgNPs display excellent stability over a period of more than six months.
When the AgNPs were left standing at room temperature without any specific protection,
no change in UV Vis absorption spectra was observed (Figure 4(a)). In addition, there
 6 S. Babu et al.
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Figure 2. TEM image of 20% green-tea-extract-capped AgNPs.

was no discernible colour change in the vials of the as-prepared AgNPs over the six months
period (Figure 4(b)). This stability could be ascribed to the presence of the deprotonated
polyphenolic compounds in the tea extract which act as both stabilising and effective
reducing agents.[32,44 46] Furthermore, UV Vis spectra of the AgNPs taken after eight
months (Figure 5) also look very similar to the UV Vis spectra of the freshly prepared

Figure 3. XRD pattern of AgNPs synthesised using 20% (V/V) tea extract
 Journal of Experimental Nanoscience 7
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Figure 4. (a) UV Vis absorption spectra of colloidal solution of AgNPs with different
concentrations of green tea leaf extract (5% 40%) after six months. (b) Visual images of the AgNPs
with different concentrations of tea leaf extracts after six months at room temperature.

nanoparticles (Figure 1). This indicates the high stability of the nanoparticles as described
above. The only difference that we observed was a slight shift in the maximum wavelength
towards the red spectrum.
However, in the presence of 100 mM NaCl, a significant decrease in the plasmon
resonance band centred at 410 nm (Figure 6) was observed. The addition of the salty
solution also induced a noticeable colour change from brown to grey of the colloidal
AgNP solution (data not shown). This indicates faster aggregation of the AgNPs in a
strong salty medium. Nevertheless, even here, this occurred after 24 h in the salty solution.

Figure 5. (a) UV Vis absorption spectra of colloidal solution of AgNPs with different
concentrations of green tea leaf extract (5% 40%) in eight months. (b) Visual images of the AgNPs
with different concentrations of tea leaf extracts in eight months at room temperature.
 8 S. Babu et al.
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Figure 6. UV Vis absorption spectra of colloidal solution of AgNPs in the presence of 100 mM
NaCl with different concentrations of green tea leaf extract (5% 40%); the stability was measured
after 24 h. (Note the significant decrease in absorbance at 410 nm.)

3.3. Detection of cysteine using AgNPs

The effect of cysteine on the freshly prepared AgNPs was studied. As shown in Figure 7(a),
the addition of increasing amounts of cysteine to the aqueous solutions of the AgNPs

Figure 7. (a) Absorption spectra of AgNPs upon addition of increasing amount of L-cysteine
(0 60 mM). Arrows indicate spectral changes with increasing concentration of L-cysteine.
(b) Calibration curve for the detection of L-cysteine (0 60 mM).
 Journal of Experimental Nanoscience 9

caused a significant decrease of the absorption band at 406 nm while a new absorption
peak appeared at 506 nm. The addition of cysteine also induced a distinct colour change
from yellow to pink that enabled the colourimetric detection of cysteine (Figure 8(b)). It is
well known that thiol groups of cysteine interact strongly with AgNPs and are easily
immobilised on the surface of the AgNPs through the formation of Ag S bonds.[50]
Hence, the resulting change in colour and shift in the wavelength clearly indicated the
cysteine-mediated aggregation of the colloidal AgNPs through hydrogen bonding and
electrostatic interactions.
The selectivity of the AgNPs for cysteine was further evaluated by monitoring the
changes in the ratio of the absorbance at 506 and 406 nm, R (A506/A406). As shown in
Figure 7(b), a good linear relationship exists between the ratio of A506/A406 and the
concentration of cysteine in the 0.0 60 mM range. It is clearly seen that the absorbance
was dependent on the quantity of cysteine added. The new absorption band at 506 nm
became more intense with the increasing concentration of cysteine. Additionally, the
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minimum detectable concentration of cysteine based on the naked-eye evaluation of

the colour change corresponded to an approximate concentration of 50 mM. The
corresponding colour variation indicates that tea-leaf-capped AgNPs can potentially
be used for the detection of cysteine through UV Vis spectroscopy or by naked-eye
observation.
The detection of cysteine by the AgNPs was also investigated in the presence of 70 mM
NaCl (Figure 9). As shown in Figure 9, although the detection of the cysteine was also
possible in the presence of 70 mM NaCl, the change in the absorbance was not as
significant as without NaCl. This probably shows that in the presence of NaCl, the AgNPs
aggregate rapidly, potentially minimising their effectiveness.

Figure 8. (Colour online) (a) UV Vis absorption spectra of AgNPs in the absence (green colour)
and presence (pink colour) of L-cysteine. (b) Visual images of the AgNPs with or without L-cysteine.
 10 S. Babu et al.
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Figure 9. UV Vis absorption spectra of AgNPs upon addition of increasing amount of L-cysteine
(0 60 mM) in the presence of 70 mM of NaCl. Arrows indicate spectral changes with an increasing
concentration of L-cysteine.

3.4. Specificity of the cysteine probe

The interference of other relative amino acids and biomolecules on monitoring cysteine
was investigated (Figure 10). The results clearly show that the synthesised AgNPs possess
high selectivity towards cysteine in the presence of other amino acids. Notably, little
spectral changes occurred in the presence of other amino acids and biomolecules. In
addition, other amino acids had no effect on the colour of the AgNPs. Therefore, the
results showed that the AgNPs can be used as simple probes for visualising the presence of
cysteine selectively over other amino acids at room temperature. Remarkably, even the
presence of methionine and histidine amino acids showed no immediate significant colour
or absorbance changes upon addition to the colloidal AgNP solution.

4. Conclusions
In summary, colloidal silver nanoparticles can be prepared by a green synthetic approach,
which involves reduction of silver nitrate using green tea extract under alkaline conditions.
The as-prepared AgNPs exhibit high stability and interesting optical properties. The as-
prepared AgNPs could be utilised as simple colourimetric and optical probes for the
selective detection of cysteine over other amino acids including methionine and histidine.
This selective detection occurs due to the specific cysteine-induced aggregation of the
AgNPs through the Ag S covalent bond formation and electrostatic interaction between
the COO¡ and NH4C zwitter ions and the thiol groups of cysteine.
 Journal of Experimental Nanoscience 11
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Figure 10. UV Vis absorption of AgNPs upon addition of 200 mM of various amino acids in the
presence of 70 mM of NaCl. The absorbance was recorded individually after the addition of each
analyte: (1) L-arginine, (2) L-proline, (3) L-alanine, (4) L-methionine, (5) L-cysteine, (6) L-valine,
(7) L-histidine, (8) L-theorenine, (9) L-iso-leucine, (10) L-leucine, (11) D/L-serine, (12) ascorbic acid,
(13) L-glutamic acid, (14) glycine, (15) urea, and (16) D-glucose.

Acknowledgments
We thank Dr. Wei Cao for the collection of TEM data, and Dr. Silvina Pagola for the XRD data
collection and analysis.

Disclosure statement
The authors declare no competing financial interest.

Funding
This work was supported by the National Science Foundation-Faculty Early Career Development
(CAREER) Award [grant number CHE-1230357] and ACE Implementation Award NSF HRD
[grant number 1238838].

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