NIR focus on energy molecule

Organisms use ATP as a universal energy storage molecule, now carbon nanotubes, modified with luciferase, have been used as near-infrared detectors for cellular ATP. The work has potential for studies of ischaemia, Parkinson's disease, hypoglycaemia and more.

Jong-HoKim, Jin-Ho Ahn, Paul Barone, Hong Jin, Jingqing Zhang, Daniel Heller, and Michael Strano of Massachusetts Institute of Technology, in Cambridge, USA, explain that all living cells need fuel to function. Adenosine triphosphate (ATP) is the cellular power supply of evolutionary choice. Because this is the case detection of this universal energy storage molecule can be used to detect bacterial, because microorganisms utilise ATP. It also represents a key subject in studies of the energetic processes of cell physiology, including ion-channel regulation and intercellular signalling cascades. From a medical perspective, ATP depletion is related to various health problems.

"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," the researchers say. MIT's Strano and colleagues have now developed just such a sensitive detection method that is both high-resolution and more robust than earlier ATP analytical techniques.

ATP is commonly detected using an assay based on the luminescence enzyme luciferase, from fireflies and other bioluminescent organisms. The assay works by using oxygen and magnesium ions to convert the enzyme's substrate luciferin into oxyluciferin, which then reacts further to produce light. The specific luciferase in question requires ATP for the light-generating process. Unfortunately, luciferase assays are relatively complicated to perform as well as time-consuming because they require the production of vectors and cell transfection methods. They also suffer from a poor signal-to-noise ratio.

The MIT team has now turned to nanotechnology to improve the luciferase protocol. They attached the luciferase to single-walled carbon nanotubes (SWNTs), which are taken up by cells relatively easily. In the presence of luciferin and ATP, oxyluciferin is formed as usual, which causes fluorescence. Crucially, however, while the carbon nanotubes would normally fluoresce in the near-infrared region of the spectrum, fluorescence at these wavelengths is extinguished proportionately by the addition of ATP to the luciferase reaction.

Strano explains further: "As it is formed, the product oxyluciferin attaches itself firmly to the nanotube," he says, "Electrons are transferred from the nanotube to the oxyluciferin so that the carbon nanotube itself can no longer fluoresce." The reduction in NIR fluorescence can easily be detected and so serves as a simple indicator of the ATP concentration. "Our new sensor is very selective for ATP," adds Strano. "We were able to use it to observe the change in ATP concentration over time and space in a 'HeLa' cell culture." The team points out that the sensor is not affected by molecules related to ATP such as adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), cytidine 5'-triphosphate (CTP), and guanosine 5'-triphosphate (GTP), which are common interferences in other ATP detection systems. The NIR fluorescence of the SWNT is only attenuated by the presence of ATP.

"The principal contribution of this work is the demonstration of a new optical sensing mechanism: enzymatic generation of a fluorescence quencher on the SWNT using a precursor that serves as the analyte," the team concludes. This is the first SWNT-based optical sensor for the detection of ATP in living cells, which also incorporates the possibility of spatial discrimination in cellular studies.