(582w) Copper Nanocluster Based Fluorescent Sensors for Sensitive and Selective Detection of Kojic Acid in Food Stuff

Kojic
acid (KA), a fungal metabolic product, has been widely used for its
preservative actions against both chemical and microbial degradation [Bentley, 2006]. However, safety
assessment for KA remains controversial and it is restricted for use by the US
Food and Drug Administration for its potential carcinogenicity, embryotoxicity and
teratogenicity [Burdock et al., 2001]. Hence,
it is highly desirable to develop highly sensitive and selective sensing systems
for KA.

In
this work, we utilized BSA-capped copper nanoclusters (CuNCs) to develop a new
fluorometric method for the determination of kojic acid with high sensitivity
and selectivity. The as-proposed biosensor was sensitive for the detection of KA.The
linear range for KA was 0.2¨C50 µM. The detection limit can be as low as 0.07 µM.
Several real foodstuff samples spiked with KA, including sauce and vinegar are analyzed
using the sensing system, and experimental results show that this fluorescent
sensor exhibits excellent recoveries. The concept of our fluorometric sensing
is illustrated in Scheme 1.

Scheme 1. Schematic
representation of the CuNCs-based sensor for kojic acid.

(1) Firstly, BSA capped
CuNCs were prepared and purified.The aqueous solution of the CuNCs exhibited violet
fluorescence under UV light. After addition of KA, the original fluorescence of
the CuNCs became weak. The corresponding fluorescence spectrum showed that the
maximum emission peak at 407 nm disappeared (Figure 1A). The fluorescence
intensity at 407 nm was plotted as a function of KA concentration and the value
decreased gradually with increasing amount of KA until a plateau was reached (Figure
1B). A good linear relationship between the fluorescence intensity and KA concentration
over the range of 0.2¨C50 µM (Figure 1B, inset) was obtained, with a detection limit
of 0.07 µM. For an excellent sensor, high selectivity is a matter of necessity.
To investigate whether our system is specific for KA, interference of various coexisting
substance in the determination of kojic acid was investigated (Figure 1C).

Figure
1. (A) Fluorescence emission spectra of the CuNCs in the presence of increasing
concentrations of KA (0¨C1000 µM). (B) Plots of the fluorescence quenching rate
at 407 nm as a function of the KA concentration (0¨C1000 µM). Inset is the linear
range for KA. (C) The responses of interfering substance to CuNCs.

(2) In
this system, several factors that may affect the quenching efficiency of KA were
optimized, such as the reaction time, pH value, buffer system, and temperature.
To understand the response rate of the fluorescence signal of the CuNCs to KA, the
time-dependent fluorescence intensity upon addition of 150 µM KA was monitored.
When KA was added to the CuNCs solution, the fluorescence intensity at 407 nm
decreased rapidly within 3 min and changed little over time. Although the
fluorescence intensity of CuNCs was dependent on temperature, the quenching
rate of KA
was
not influenced. This process was notable as it involved green chemistry, simple,
stable, fast and highly selective.

(3) Furthermore,
the mechanism of the sensitive fluorescence response of BSA-CuNCs toward KA has
also been investigated. It is observed that the surface of the clusters is stabilized
by a small amount of Cu²⁺, which should have strong and specific
interactions with KA. The selective binding of KA to copper ions promotes the
formation of copper kojate precipitation [Barham, 1939] on the surface
of CuNCs, and thus to statically quench the fluorescence of CuNCs as given in
scheme 1. A new peak at 314 nm appeared on the absorption spectra of CuNCs in
the presence of increasing concentrations of KA, which proves the formation of
a new complex, namely
copper
kojate in this case. Additionally, the excited state lifetime of the BSA-CuNCs in
the absence and presence of KA was compared (2.454 ns and 2.416 ns,
respectively). The result showed that the lifetime of the CuNCs hardly changed with
the addition of KA, further implying
static quenching. Based on these results, we concluded that the mechanism for
the fluorescence quenching behavior was a static quenching process.

This work was supported by the Natural Science Foundation of China
(Nos 21276192, 51173128 and 31071509), the Ministry of Science and Technology of
China (Nos 2012YQ090194, 2012BAD29B05 and 2012AA06A303), and the Ministry of
Education (No. NCET-11-0372).

 References

1.       Barham,
H. Industrial & Engineering Chemistry Analytical Edition, 11, 31-33,1939

2.       Bentley,
R. Natural Product Reports, 23, 1046-1062,2006

3.       Burdock,
G.A., Soni, M.G., Carabin, I.G. Regulatory Toxicology and Pharmacology, 33,
80-101,2001