A Luciferase/Single‐Walled Carbon Nanotube Conjugate for Near‐Infrared Fluorescent Detection of Cellular ATP
A Luciferase/Single‐Walled Carbon Nanotube Conjugate for Near‐Infrared Fluorescent Detection of Cellular ATP
A plain-language reading has not been prepared for this paper yet.
Original abstract
All micro-organisms use adenosine 5’-triphosphate (ATP) as a universal energy storage molecule, and thus knowledge of its concentration is central to the detection of bacterial contamination and the study of energetic processes in cell physiology from ion-channel regulation to intercellular signaling cascades. Additionally, ATP depletion is related to pathogenesis such as ischemia, Parkinson s disease, and hypoglycemia. There remains a persistent need for more sensitive, higher-resolution, and more robust detection of ATP for, among other goals, the understanding of its spatial compartmentalization within living cells. For this purpose, the conventional method of ATP assay within living cells is luciferase(Luc)-mediated bioluminescence, whereby ATP reacts at the enzyme in the presence of d-luciferin (Lrin) and Mg to produce oxyluciferin (oxyLrin) and a fluorescent emission. However, this approach, which involves synthesis of Luc vectors and cell transfection is tedious, timeconsuming, and has a low signal-to-noise ratio. The extension of this method to the modulation of quantum confined nanorods or nanotube fluorophores, such as single-walled carbon nanotubes (SWNT), has not been addressed to date, despite obvious benefits in sensitivity and photobleaching resistance. Herein, we report a SWNT/Luc enzyme conjugate (SWNT) in which the bioluminescent reaction selectively recognizes ATP at luciferase. The SWNT near-infrared (NIR) fluorescence is ultimately quenched by a two-step reaction that involves detection of a target and generation of a redox quenching intermediate. This SWNT sensor is very selective to ATP, but not to adenosine 5’-monophosphate (AMP), adenosine 5’-diphosphate (ADP), cytidine 5’-triphosphate (CTP), and guanosine 5’-triphosphate (GTP), and is also able to detect ATP temporally and spatially in living HeLa cells. The approach, whereby an enzyme–nanotube complex creates a redox quenching intermediate from the target analyte, can be extended to a wide range of biologically important analytes. We first constructed the Luc-conjugated SWNTs as shown in Figure 1 (see the Supporting Information). After immobilization of Luc on SWNTs functionalized with phospholipids