Sol-gel and polyacrylamide based optical pebble nanosensors for intracellular imaging and analysis of oxygen and glucose.

摘要

Currently, there is a lack of reliable methods for real-time, non-invasive, and quantitative intracellular measurements. Common methods for single cell analysis include nano optodes and electrodes. While the tips of these sensors may have nanometer dimensions, mechanical and physical perturbations of the cell are caused by punching holes in cellular membrane during measurements and the large sensor penetration volume when inserted into the cells. "Naked" (free) fluorescent sensing dyes have also been widely used as intracellular indicators. These individual molecular probes are physically minute but usually suffer from the chemical interference between probe and cellular components, such as protein interferences and sequestration. Also, the free dye itself may be toxic to the cell or to specific cellular organelles.; A recent development in sensor design combines the advantages of sensor tips and molecular probes, i.e. simultaneously avoids both physical and chemical interferences between sensor (probe) and sample (cell or organelle). These spherical nanosensors or PEBBLEs (Probes Encapsulated By Biologically Localized Embedding) are optical sensors of nanometer dimensions with optical sensing components (i.e. fluorescent dyes) entrapped within an inert matrix. The small size of the PEBBLE sensors enables them to be inserted non-invasively into a living cell, and the sensor matrix protects the cell from the cyto-toxicity of sensing components and the sensing components from the cellular interferences. Due to these superior properties, PEBBLE sensors provide a more reliable optical method for non-invasive and quantitative intracellular measurements.; In this body of work, two matrices, sol-gel silica and polyacrylamide, were used for the fabrication of PEBBLE sensors designed for intracellular oxygen and glucose measurements. These sensors range from ca. 50--350 nm in size, and are produced by dispersion and emulsion fabrication techniques. They usually contain a fluorescent indicator dye together with a reference dye for the purpose of ratiometric measurements. The characterizations of these sensors in terms of linear range, reversibility, photostabillity, dye leaching, interference study, response time and ratiometric validation, as well as their intracellular and biomedical applications, are described. Additionally, an appendix describes the use of such probes (sensitive to oxygen) for in-vitro demonstration of photodynamic therapy.