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