Using Gold Nanoclusters As Selective Luminescent Probes for Phosphate-Containing Metabolites Po-Han Li, Ju-Yu Lin, Cheng-Tai Chen, Wei-Ru Ciou, Po-Han Chan, Liyang Luo, Hung-Yu Hsu, Eric Wei-Guang Diau, and Yu-Chie Chen* Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan * S Supporting Information ABSTRACT: Glutathione-bound gold nanoclusters (AuNCs@GSH) can emit reddish photoluminescence under illumination of ultraviolet light. The luminescence of the AuNCs@GSH is quenched when chelating with iron ions (AuNCs@GSH-Fe 3+ ), presumably resulting from the effective electron transfer between the nanoclusters and iron ions. Nevertheless, we found that the luminescence of the gold nanoclusters can be restored in the presence of phosphate- containing molecules, which suggested the possibility of using AuNCs@GSH-Fe 3+ complexes as the selective luminescent switches for phosphate-containing metabolites. Phosphate- containing metabolites such as adenosine-5′-triphosphate (ATP) and pyrophosphate play an important role in biological systems. In this study, we demonstrated that the luminescence of the AuNCs@GSH-Fe 3+ is switched-on when mixing with ATP and pyrophosphate, which can readily be observed by the naked eye. It results from the high formation constants between phosphates and iron ions. When employing fluorescence spectroscopy as the detection tool, quantitative analysis for phosphate- containing metabolites such as ATP and pyrophosphate can be conducted. The linear range for ATP and pyrophosphate is 50 μM to sub-millimolar, while the limit of detection for ATP and pyrophosphate are ∼43 and ∼28 μM, respectively. Additionally, we demonstrated that the luminescence of the AuNCs@GSH-Fe 3+ can also be turned on in the presence of phosphate- containing metabolites from cell lysates and blood plasma. M etabolites play an important role in biological systems. For example, adenosine-5′-triphosphate (ATP) is the main energy carrier in cells 1 and also acts as an extracellular signaling agent in many biological processes. 2,3 Pyrophosphate is generated when ATP is hydrolyzed into adenosine monophosphate (AMP). The intracellular concentration of pyrophosphate is ∼50 μM, while the extracellular concentration of pyrophosphate is ∼3 μM. 4 In general, the concentration of ATP in cells is at the millimolar level, which is much higher than those of other phosphate-containing metabolites, such as pyrophosphate, adenosine diphosphate (ADP), and AMP. 4,5 It has been known that the level of phosphate-containing metabolites in cells can be used as an indicator of dysfunctions or disorders in biological systems. 6,7 The level of ATP has been used in determination of cell viability. 8 Additionally, the physiologic concentration of blood phosphates in healthy individuals is also at the millimolar level, 9 which is mainly dominated by ATP. 4 Because hypophosphatemia and hyper- phosphatemia may result in illness, the phosphate level in blood can be used as an indication for health conditions of individuals. 9 Thus, the development of analytical methods is essential in indicating the level of phosphate-containing metabolites in biological samples. Traditional ATP assays such as the luciferase assay have been widely used in the determination of ATP levels in vitro. 10-12 Although the method is sensitive, expensive enzymes and substrates are required for the assays. Thus, efforts have been devoted in designing suitable probe molecules as the sensing probes for ATP 13-15 and pyrophosphate. 16-18 For example, an organic probe composed of a pincer-like benzene-bridge with a pyrene excimer as a signal source and imidazolium as a phosphate anion receptor has been successfully demonstrated as an efficient ATP sensor, 14 while coumarin derivatives have been designed as highly selective fluorescent sensors for pyro- phosphate. 18 Electrochemistry 19-21 and spectroscopy 22-33 are commonly employed as analytical tools for the detection of ATP and pyrophosphate. In addition to organic probes, nanoparticles have been used as probes to facilitate the detection process. 26-28 Sensing interactions involve aptamer- based molecular recognition 22-26 and metal ion-based chelation. 29-33 Glutathione (GSH) is a tripeptide consisting of N-γ- glutamyl-cysteinyl-glycine and is used as a reducing agent 34-41 for the generation of gold nanoclusters, which has a maximum luminescence at ∼613 nm under an excitation of the light at 396 nm (Figure S1 in the Supporting Information). Received: February 2, 2012 Accepted: May 31, 2012 Published: June 11, 2012 Technical Note pubs.acs.org/ac © 2012 American Chemical Society 5484 dx.doi.org/10.1021/ac300332t | Anal. Chem. 2012, 84, 5484-5488