In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets Ying Wang, †,‡,⊥ Zhaohui Li, ‡,§,⊥ Thomas J. Weber, ‡ Dehong Hu, ‡ Chiann-Tso Lin, ‡ Jinghong Li,* ,† and Yuehe Lin* ,‡ † Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, China 100084 ‡ Pacific Northwest National Laboratory, Richland, Washington 99352, United States § College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, China 450001 * S Supporting Information ABSTRACT: Nucleotides, for example, adenosine-5′-triphos- phate (ATP) and guanosine-5′-triphosphate (GTP), are primary energy resources for numerous reactions in organisms including microtubule assembly, insulin secretion, ion channel regulation, and so on. In order to advance our understanding of the production and consumption of nucleoside triphos- phates, a versatile sensing platform for simultaneous visual- ization of ATP, GTP, adenosine derivates, and guanosine derivates in living cells has been built up in the present work based on graphene oxide nanosheets (GO-nS) and DNA/RNA aptamers. Taking advantage of the robust fluorescence quenching ability, unique adsorption for single-strand DNA/RNA probes, and efficient intracellular transport capacity of GO-nS, selective and sensitive visualization of multiple nucleoside triphosphates in living cells is successfully realized with the designed aptamer/GO-nS sensing platform. Moreover, GO-nS displays good biocompatibility to living cells and high protecting ability for DNA/RNA probes from enzymatic cleavage. These results demonstrate that the aptamers/GO-nS-based sensing platform is capable of selective, simultaneous, and in situ detection of multiple nucleotides, which hold a great potential for analyzing other biomolecules in living cells. N ucleotides, consisting of a nucleobase and a five-carbon sugar as well as one phosphate group, are mainly considered as the basic building block units in nucleic acids. However, some nucleotides also have many other important roles in metabolism and in metabolic control. Among them, adenosine-5′-triphosphate (ATP) and guanosine-5′-triphos- phate (GTP) are found as typical energy molecules regulating various biological processes. 1,2 As the primary energy molecule in living cells, ATP is generally called as the “molecular unit of currency” for intracellular energy transfer, 3-5 which is highly necessary for some biochemical reactions such as muscle contraction, biomolecule synthesis and degradation, membrane transportation, and signal transduction, etc. 6-9 Meanwhile, GTP plays important roles in protein synthesis and holds great significance for signal transduction in living cells. 10-13 Most importantly, ATP and GTP could act coordinately to realize numerous reactions such as microtubule assembly, insulin secretion, and ion channel regulation. 14-17 Therefore, the analysis, especially in situ simultaneous visualization of ATP and GTP, has great importance to advance our understanding of their behavior, function, and interaction inside living cells. 18-21 In the past decades, numerous attempts have been made to realize the detection of either ATP or GTP. For example, biosensors based on fluorescent molecules like acridine, polythiophene, or imidazolium anthracene derivate have been used for ATP detection. 22-24 Luciferase (an ATP-consuming enzyme) and lymphoid ecto-adenylate kinase have been combined to measure cellular ATP levels in some cases. In addition, ATP aptamer sensors making use of fluorescent, electrochemical, and colorimetric methods have been reported in previous studies. 25,26 Similarly, synthesized fluorescent dyes, such as water-soluble imidazolium anthracene derivative and benzimidazolium with unique specificity, have been applied for GTP detection in buffer solutions as well as biological fluids. 19,21,27 However, most of the assays could only detect ATP or GTP, respectively. Lack of methods is an obstacle to realize the simultaneous detection of ATP and GTP inside living cells. Accordingly, a suitable in situ analyzing assay for multiple biotargets in living cells is highly desirable. In recent years, graphene oxide (GO) has been emerging with several unique properties including planar sheet structure, fluorescence quenching ability, easy functionalization, and good biocompatibility. 28-32 For example, GO has been utilized for the ultrasensitive detection of cyclin A 2 with a detection limit of Received: March 22, 2013 Accepted: June 11, 2013 Published: June 11, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 6775 dx.doi.org/10.1021/ac400858g | Anal. Chem. 2013, 85, 6775-6782