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ScienceDirect
Energy Procedia 56 (2014) 10 – 18

11th Eco-Energy and Materials Science and Engineering (11th EMSES)

Gold nanoparticles-based colorimetric sensor for

cysteine detection

Suriyapha Jongjinakool, Khwankhao Palasak, Natvara Bousod, Siriwan Teepoo*

0F

Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathumthani
12110, Thailand

Abstract

A simple, sensitive and selective colorimetric method for the detection of cysteine was demonstrated with unmodified gold
nanoparticles (AuNPs) as probes. In this approach, the synthesized AuNPs solution was stabilized by the citrate anions as their
repulsion protected the AuNPs from aggregation. Cysteine was added to AuNPs solution and was incubated to react for 3 min.
The resulting mixture color changes dramatically from red-purple-blue because cysteine induced the nanoparticle aggregation.
These processes were studied and characterized by UV–vis spectroscopy, zeta potential and dynamic light scattering. Several
parameters including AuNPs size, reaction time and media pH that governed the analytical performance of the method have been
studied in detail and optimized. Under the optimized experimental conditions, cysteine could be selectively detected in a
concentration range from 0.1 to 0.6 ppm with a limit of detection as 0.01 ppm at a signal-to-noise ratio of 3. The sensitivity was
calculated as 1.474 Abs/ppm. Some common interferents such as Na+, Cu2+, Cl- and urea showed no interference in the
determination of cysteine by using AuNPs.

©2014
© 2014Elsevier
The Authors. Published
Ltd. This is an openbyaccess
Elsevier Ltd.
article under the CC BY-NC-ND license
Peer-review under responsibility of COE of Sustainalble Energy System, Rajamangala University of Technology Thanyaburi
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
(RMUTT). under responsibility of COE of Sustainalble Energy System, Rajamangala University of Technology Thanyaburi (RMUTT)
Peer-review

Keywords: Cysteine; Gold nanoparticle; Colorimetric; Aggregation

Corresponding author at: Tel: +66-02-5493535; Fax: +66-02-5493526

E-mail addreass: siriwan@mail.rmutt.ac.th.

1876-6102 © 2014 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of COE of Sustainalble Energy System, Rajamangala University of Technology Thanyaburi (RMUTT)
doi:10.1016/j.egypro.2014.07.126
 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18 11

1. Introduction

In the past decades, there has been rapid growth in nanotechnology research. Nanotechnology deals with
processes that take place on the nanometer scale, that is, from approximately 1 to 100 nm. In nanotechnology, the
assembly of metallic nanoparticles has resulted in novel materials with interesting electronic, optical and chemical
properties [1-3]. Metal nanoparticles have attracted great interests as the both chemical sensor and biosensor because
of their easy biofunctionalization, high surface area and spectral properties [4]. Among these metal nanoparticles,
gold nanoparticles (AuNPs) have found useful applications in chemical analysis [5-7]. In recent few years the use
of AuNPs as sensing probe for the detection of important analyses base on aggregation of nanoparticles was
dramatically increased.
AuNPs can exhibit unusual chemical, physical, electrical and optical properties that are not likely in bulk
materials. The optical properties of AuNPs have attracted scientists because of their applications as a colorimetric
probe [8-10]. AuNPs as colorimetric sensor or probe have been widely used for several analytes such as mutations
[8], immunoglobulin G [11], mercury(II) ion [12], cartap [13] and melanine [14]. AuNPs provided high sensitivity
for the detection because they exhibit characteristic surface plasmon resonance (SPR) absorption properties. The
resonance frequency of this SPR is strongly dependent upon the size, shape, dielectric properties, and local
environment of the nanoparticles [15]. In most of the cases the use of AuNPs as chemical sensing can achieved by
mornitoring the changes in the color upon aggregation/dissociation processes. The color of AuNPs may range from
red to purple, to blue and almost black, due to the formation of aggregates. In colloidal solutions, AuNPs are red in
color because of the Mie absorption by their surface plasmon oscillation that peaks at 520 nm [16]. AuNPs have
been induced to aggregate by the addition of molecule which presented of amine or thiol groups via non-covalent
bonding. The aggregation of AuNPs leads to the formation of a new absorption band at longer wavelengths as a
result of color change from red to purple-blue depending on their particle size [17].
Cysteine is a non-essential Į-amino acid containing nonpolar sulphydryl (-SH) group that provides its
participation in a variety of biochemical reactions. The lack of cysteine is responsible for many kinds of different
diseases. It causes slow hair growth, depigmentation, damage of liver and muscles [18]. Therefore, the monitoring
of cysteine in biological matrices is highly demanded. A number of methods have been developed for the assay of
cysteine such as spectrofluorimetric [19], chemiluminescent [20], electrochemistric [21] and chromatographic
methods [22]. These methods are generally laborious and time consuming. In addition, procedures require expensive
and complicated instrumentation that make them unattractive to routine analysis.
Thus, this work investigated the development of method for cysteine detection with cost effective, short
analysis time and no requirement of expensive instrumentation. A propose method was evaluated by using
unmodified AuNPs. The cysteine presented -NH2 and -SH which offered to coordinate to AuNPs and cross-link
AuNPs causing aggregation. The clearly distinguish able color change facilitates a simple sensor was developed for
cysteine detection. Hence, in this work we gave the demonstration of the colorimetric detection of cysteine using
unmodified AuNPs as probes. The assay procedure for the colorimetric detection of the cysteine is illustrated in
scheme 1.

Added cysteine Added cysteine

0.1 ppm 0.6 ppm

AuNP dispersed AuNP aggregated AuNP aggregated

Scheme 1. Schematic representation of the colorimetric assay for cysteine detection using AuNPs.
12 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18

2. Materials and methods

2.1 Materials

All chemicals used are of analytical grade. All solutions were prepared with deionized water. Chloroauric
acid (HAuCl4), trisodium citrate and sodium borohydride (NaBH4) were purchase from Aldrich-Sigma. Cysteine
was obtained from Hi Media Laboratories Pvt.Ltd.

2.2. Instrumentation

UV–vis spectroscopic measurement was performed by a UV 1601 spectrophotometer (Shimadzu, Japan) using
a 1 cm of quartz cuvette. The spectrum of solution in the range of 300–800 nm was recorded. All the experiments
were carried out at room temperature.

2.3 Preparation of AuNPs

Typically, all glassware were cleaned with 10% w/v HNO3, and rinsed thoroughly with deionized water prior
to use. All other solutions were prepared with deionized water. AuNPs were prepared according to the literature
[23], by adding 153 μl of HAuCl4 solution into 500 ml of trisodium citrate sodium solution. Then, 1,200 μl of
NaBH4 was rapidly added into above mixture solution, which resulted in a color change from pale yellow to finally
arrive at red–purple. The absorption maximum of the synthesized colloidal gold in the UV–vis spectrum was at 520
nm and the solution was stored in a refrigerator in a dark-colored glass bottle before use.

2.4 Preparation of AuNPs with various sizes

The particles size of nanoparticles depended on reducing agent. The procedure was carried out as same as 2.3
except the concentration of NaBH4 which act as strong reducing agent. Amount of NaBH4 was varied from 200 to
1,200 μl. The particle size of AuNPs was measured by using dynamic light scattering (DLS).

2.5 Colorimetric assays based on AuNPs aggregation

In order to determine cysteine concentration by aggregation of AuNPs, a range of concentrations of cysteine

were prepared (0.1-1.0 ppm). Each cysteine solution was added to 1,800 ȝl of AuNPs solution and the resulting
mixtures were then allowed to react for 3 min. Subsequently, the absorbance changes were monitored by UV-visible
spectrophotometry. The incubation time and pH values were investigated on the aggregation of AuNPs. All of the
experiments were done at room temperature. The determination of cysteine was observed by plotting the absorbance
at 615/520 nm vs. concentration.

3. Results and discussion

3.1 Characterization of AuNP

AuNPs were synthesized by the reduction of HAuCl4 with sodium citrate and NaBH4 in aqueous solution. The
pH value of the synthesized AuNPs was measured to be around 6.00. The zeta potential of AuNPs was -32 mV
which indicated occurring negative charge on the surface of AuNPs. The UV-vis absorption spectrum of the
colloidal solution is shown in Figure 1. The absorption band centered at 520 nm is characteristic of AuNPs and is
due to the surface plasmon resonance absorption which gives rise to the AuNPs distinctive red color.
 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18 13

1
1.0
520 nm
0.8

Absorbance
0.6

0.4

0.2

0
400 450 500 550 600 650 700 750 800

Wavelenght (nm)

Fig. 1 Absorption spectra of AuNPs dispersed in aqueous solution

3.2 The effect of reducing agent on the AuNPs size

The different particle sizes were synthezied by vary the concentrations of NaBH4. The results showed that
the the particle size decreased with increasing volume of NaBH4 ranging from 200 to 1,200 μl. The color of the
solution varies from red to pink depending on the size of the particles. The surface plasmon resonance of the gold
AuNPs is red shifted with increase in particle size in accordance to Mie theory. The particle size was varied within
the size range of 10.8 to 13.1 nm (fig. 2A–D) where the concentrations of the gold solution are same in all cases.
The size of 10.8 nm was selected for further experiments due to highest their surface area helping aggregation
process.

Fig. 2 DLS results of synthesized AuNPs using NaBH4 of 200 μl (A), 300 μl (B), 600 μl (C) and 1,200 μl (D)
14 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18

3.3 Colorimetric detection of cysteine

Scheme 1 illustrates the concept of this proposed procedure, which was based on aggregation of AuNPs.
AuNPs in solution are stabilized by adsorbed negative ions whose repulsion prevents the strong van der Waals
attraction between gold particles from causing them to aggregate. In the absence of cysteine, AuNPs were dispersed
in solution. When the standard cysteine solutions of different concentrations were added to the AuNPs dispersion,
the color changed dramatically from red-purple-blue. As shown in Fig. 3, the characteristic plasmon resonance peak
of dispersed AuNPs is at 520 nm. With increasing cysteine concentration, the intensity of the peak at 520 nm
becomes weaker while a new surface plasmon resonance band at about 615 nm appears.

Fig. 3 UV–vis spectra and visual color change obtained from adding the cysteine at 0.1 ppm (a), 0.5 ppm (b),
1.5 ppm (c) into AuNPs solution

The corresponding colorimetric effect was evaluated by comparing the extinction ratio between 615 nm and
520 nm (A615/A520) for cysteine detection.
The aggregation kinetics of this assay was studied by monitoring the A615/A520 vs time (Fig.4). The
A615/A520 values of solutions with 0.5 ppm cysteine increased gradually within 3 min and then remained constant
when elongated the time. Therefore, the aggregation of AuNPs induced by cysteine was almost complete within 3
min. The data were recorded at 3 min in each experiment.

0.74

0.72
A615/A520

0.70
0.7

0.68

0.66
0
0 11 22 33 44 55 6

Time (min)

Fig. 4 The kinetics of aggregated AuNPs by cysteine

 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18 15

pH of AuNPs solution is a key factor for aggregation. To monitor the effect of pH of the medium, the
adsorption measurements were carried out at pH values 4.00 to 10.00. The results showed that almost the absorption
ratio A615/A520 decreased with increasing pH value as fig.5. The adjustment to a pH value below 4.00 already
caused the AuNPs solution to turn blue, which showed that the AuNPs had already aggregated. Thus, pH of media
was chosen as 4.00.

Fig. 5 The effect of pH media

3.4 Study of the interference influence on aggregation

The influences of common molecules with present in general samples were also investigated such as Na+,
Cu , Cl- and urea to test the selectivity of the developed method. The selectivity of the colorimetric sensor for
2+

cysteine was carried out by monitoring the A615/A520 response in the presence of interferences. The results showed
that no color changes were observed. Thus, the propose method had good specificity and selectivity.

3.5 Calibration curve of cysteine detection

Under the above-mentioned optimized experimental conditions, a series of different concentrations of cysteine
was respectively added and their UV–vis spectra were recorded. The increasing addition of cysteine to the AuNPs
solution produced the characteristic particle aggregation and led to a red color change to purple and blue as in Fig.
6A. As mentioned above, the degree of aggregation depended on the cysteine concentration and confirmed with
DLS results. Fig. 6B showed the average diameter of AuNPs in presence of different concentrations of cysteine.
After the addition of cysteine, the diameter of the unmodified AuNPs increased from 22 to 54,717 nm. Fig. 6C
displayed the plots of the absorption ratio (A615/A620) over varying concentrations of cysteine. When increasing
the concentration of cysteine, the absorption ratio increased. A good linear relationship (r = 0.998) between
A615/A620 and cysteine concentrations from 0.1 to 0.6 ppm. A limit of detection of 0.01 ppm was estimated using
three times the standard derivation in blank solution (n=10).
16 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18

Fig. 6 (A) Photograph, (B) DLS results and (C) calibration of cysteine at different concentrations from
0.1 to 1.0 ppm
 Suriyapha Jongjinakool et al. / Energy Procedia 56 (2014) 10 – 18 17

4. Conclusion

A colorimetric method was developed to detect cysteine using unmodified AuNPs. This approach probably
relies on aggregation of AuNPs which were induced by cysteine causing color change from red to blue. The
proposed method allowed the detection concentration as low as 0.01 ppm to achieve with the naked eye within 3
min. Our strategy has various advantages including simplicity, convenience and low cost.

Acknowledgments

We would like to thank faculty of Science and Technology, Rajamangala University of Technology
Thanyaburi, for financial support. The authors also thank Polymer colloid Lab at the Department of Chemistry,
Rajamangala University of Technology Thanyaburi, for support on the DLS instrument.

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