Article pubs.acs.org/ac
Glutathione-Protected Silver Nanoclusters as Cysteine-Selective Fluorometric and Colorimetric Probe Xun Yuan,† Yuanqi Tay,† Xinyue Dou,† Zhentao Luo,† David Tai Leong,† and Jianping Xie*,† †
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore S Supporting Information *
ABSTRACT: The integration of the unique thiol-Ag chemistry and the specific steric hindrance from the organic layer of fluorescent Ag nanoclusters (AgNCs) was first developed in this work to achieve a simple detection of cysteine (Cys) with high selectivity and sensitivity. The key design is a strongly red-emitting AgNC protected by the interference biothiol, glutathione, or GSH (hereafter referred to as GSH-AgNCs), where both the physicochemical properties (Ag surface chemistry and fluorescence) of the NC core and the physical properties (e.g., steric hindrance) of the organic shell were fully utilized for Cys detection with three major features. First, owing to the specific thiol-Ag interaction, the fluorescent GSH-AgNCs showed superior selectivity for Cys over the other 19 natural amino acids (nonthiol-containing). Second, the GSH protecting layer on the NC surface made possible the differentiation of Cys from GSH (or other large-sized thiol molecules) simply by their size. Third, the ultrasmall size of GSH-AgNCs and the high affinity of the thiol-Ag interaction provided high sensitivity for Cys detection with a detection limit of 2 nm) typically used in previous studies.30,31 The sensitivity of our assay was further enhanced by the strong interaction between the thiol group and Ag atoms on the NC surface, which has high efficiency in quenching the fluorescence of
observation (via naked eye) of the Cys detection. However, the falsified signal can be easily corrected by the second modal − the colorimetric modal, where no color changes were observed for both Lys_GSH-AgNCs and Trp_GSH-AgNCs (Figure 3a, top panel). This visual observation verified that Lys and Trp did not affect the optical properties of fluorescent GSH-AgNCs. The dual-modal detection demonstrated in this study can largely minimize the falsified signals in the single detection modal (this is also a common challenge in other fluorometric assays for Cys), which could greatly facilitate the internal cross checking if needed. The steric hindrance provided by the GSH protecting layer of GSH-AgNCs also made possible the differentiation of Cys from GSH in our sensor system. The size of Cys [molecular weight (MW) of ∼121 Da] is much smaller than GSH, which is a tripeptide with a MW of 307 Da. As demonstrated in Figure 2c, the small thiol ligand (Cys) can penetrate the GSH protecting layer of AgNCs, interacting with the Ag atoms on the NC surface, leading to the decomposition and the subsequent fluorescence quenching of GSH-AgNCs. In contrast, the steric hindrance from the GSH protecting layer can effectively prevent the penetration and subsequent thiol etching of bulky thiol ligands. This hypothesis was affirmed by several revealing experimental observations. As shown in Figure 4a, the addition of GSH or other large thiol molecules (e.g., BSA, MW of 67 kDa) to the red-emitting GSH-AgNCs has 1917
dx.doi.org/10.1021/ac3033678 | Anal. Chem. 2013, 85, 1913−1919
Analytical Chemistry
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GSH-AgNCs. It is therefore possible that one Cys molecule can completely quench the fluorescence of one GSH-AgNC via the removal of Ag atoms from the NC surface and decomposing the fluorescent NCs into small and nonfluorescent complex species. This feature is difficult or impossible to be achieved in sensors using large NPs. To evaluate the sensitivity of our assay, different concentrations of Cys (0−1 μM) were spiked with red-emitting GSH-AgNC solution (100 nM). As shown in Figure 5, the fluorescence of GSH-AgNCs was gradually decreased when the Cys concentration was increased from 0 to 1 μM (Figure 5a). A linear decrease in fluorescence intensity was observed in the Cys concentration range of 0−500 nM (Figure 5b). The limit of detection (LOD) for Cys at a signalto-noise ratio of 3 was estimated to be 3 nM, which are 2 orders of magnitude lower than the LOD reported in other NPs-based Cys sensors.30 It is worth mentioning that the LOD of our assay is also determined by the concentration of GSH-AgNCs in the sensor construction, where a lower concentration of GSH-AgNCs should achieve a lower LOD for Cys detection. Therefore, we could even achieve a LOD in the pM range by using very low concentration of GSH-AgNCs (e.g., 1 nM). The versatility of the detection regime could further broaden the usability of our assay for samples in various practical settings where the Cys concentration may vary widely.
AUTHOR INFORMATION
Corresponding Author
*Phone: +65 6516 1067. Fax: +65 6779 1936. E-mail: chexiej@ nus.edu.sg. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by the Ministry of Education, Singapore, under Grants R-279-000-295-133 and R-279-000327-112. X. Yuan acknowledges the National University of Singapore for his research scholarship.
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CONCLUSIONS In summary, we have developed a new and simple (in sensor construction and operation) method to detect Cys with high selectivity and sensitivity using highly fluorescent AgNCs. The unique thiol-Ag chemistry and the specific steric hindrance from the organic layer of the NCs were integrated into one single optical-active material (GSH-AgNCs) in this study, where both physicochemical properties (Ag surface chemistry and fluorescence) of the NC core and physical properties (e.g., steric hindrance) of the organic shell of GSH-AgNCs were fully utilized for Cys detection. Owing to the unique and strong thiol-Ag interaction, the fluorescent GSH-AgNCs showed superior selectivity for Cys over the other 19 natural amino acids (nonthiol-containing). The unique design of the GSH layer on the NC surface, however, made possible the differentiation of Cys from GSH (or other large-sized thiol molecules) simply by their size. Furthermore, the ultrasmall size of GSH-AgNCs and the high affinity of thiol-Ag interaction provided high sensitivity for Cys detection with a LOD of
