PEBBLE Nanosensors for Intracellular Imaging and Analysis of Free Calcium and Zinc.

[摘要] Ca2+ is a universal second messenger and plays a major role in intracellular signaling, metabolism and a wide range of cellular processes. To date, the most successful approach for intracellular Ca2+ measurement involves the introduction of optically sensitive Ca2+ indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca2+ indicators for intracellular sensing and imaging has several limitations, such as interference from cellular small ions and biomacromolecules, and unwanted sequestration of the indicator molecules. Zinc is the second most abundant trace element in the human body. It is an essential component of all six classes of enzymes and several families of regulatory proteins. Zinc deficiencies and excesses were found to be related to a number of health issues. Due to the importance of zinc in the human body, many fluorescent Zn2+ indicators have been developed. Among these indicators, carbonic anhydrase (CA) and its mutants are particularly useful for intracellular Zn2+ sensing, because of their outstanding and tunable sensitivity (picomolar level), selectivity and binding kinetics.In this work, PEBBLE nanosensors have been developed for intracellular measurements of free Ca2+ and Zn2+. The general design, matrices and the advantages of PEBBLE nanosensors are briefly reviewed in Chapter 1. Chapter 2 describes the preparation, characterization and intracellular application of PEBBLE nanosensors encapsulated with rhodamine based Ca2+ fluorescence indicators. The Kd of Rhod-2 inside PEBBLEs is determined to be 500~600 nM for in-solution calibration and 1 µM for in-cell calibration. Chapter 3 describes the preparation and purification of CA encapsulated PEBBLEs; the active CA encapsulation is about 28%. Chapter 4 describes zinc sensing performed by CA encapsulated and conjugated PEBBLEs. The Zn2+ binding affinity of the CA conjugated on PEBBLE surface is determined to be 9 pM, while the Zn2+ sensing by CA encapsulated PEBBLEs is not successful. Chapter 5 summarizes the conclusions and limitations of this work, and proposes future experiments for developing novel PEBBLE nanosensors.

[发布机构] University of Michigan

[效力级别] Calcium Ion

[关键词] Nanoparticles;Calcium Ion;Zinc Ion;Carbonic Anhydrase;Chemistry;Science;Chemistry