Direct electrochemical observation of glucosidase activity in isolated single lysosomes from a living cell

Abstract

SignificanceThe quantification of protein activity in individual lysosomes in living cells is realized using a nanocapillary designed to electrochemically analyze internal solution, in which a single lysosome is sorted from the cell and the target protein is reacted with the corresponding kit components to generate hydrogen peroxide for measurement. The ability to sort and assay multiple lysosomes from the same cell allows direct study of protein function at subcellular resolution and provides unprecedented information about the homogeneity within the lysosomal population of a single cell.

Ultrasmall Nanopipette: Toward Continuous Monitoring of Redox Metabolism at Subcellular Level

“…[1] Theevolution of aerobic metabolism led to the production of reactive oxygen species (ROS), which are recognized as the most important messengers in redox signaling pathways. [1] Theevolution of aerobic metabolism led to the production of reactive oxygen species (ROS), which are recognized as the most important messengers in redox signaling pathways.…”

“…The monitoring of redox metabolism in single living cell is of great importance to revealing insights into heterogeneous cellular features,a sw ell as studying how cells deal with environmental stresses at the molecular level to gain am ore dynamic understanding of cellular functions. [1] Theevolution of aerobic metabolism led to the production of reactive oxygen species (ROS), which are recognized as the most important messengers in redox signaling pathways. [2] Toward elucidating the complex biological roles of ROS,i ti sc ritical to develop analytical technologies to monitor their dynamic changes within localized regions of the cell.…”

Intracellular Wireless Analysis of Single Cells by Bipolar Electrochemiluminescence Confined in a Nanopipette

“…Following our previously developed nanokit, [28,29] the addition of enzymes in the pipette could convert the corresponding molecules into hydrogen peroxide for the…”

“…Simultaneously, the cytosol is electrochemically sorted from the outside to the inside compartment of the nanopipette through the nanochannels of this porous Pt deposit. [27][28][29] Under this electric field, the intracellular molecules reacted with the corresponding reagents including L012 (a luminol analogue with high ECL efficiency) [30] inside the nanopipette and produced bipolar ECL emission as the optical readout [31,32] [reactions (1) and (2)]:…”

Harpagide, a natural product, promotes synaptic vesicle release as measured by nanoelectrode amperometry

“…high spatiotemporal resolution, high sensitivity, minimum damage to cells) [31][32][33][34][35][36] in real time monitoring of molecular events at multiple-scale (cell populations, single cell and subcellular) levels. [37][38][39][40][41][42][43][44][45][46][47][48] Further combining with scanning electrochemical microscopy (SECM) or scanning ion conductance microscopy (SICM) allows electrochemical and topographical imaging of neurons and detection of neurotransmitter release with super resolution. [49][50][51] Recently, amperometry at carbon-based nanoelectrodes has demonstrated its unique advantages for monitoring in real time individual vesicular exocytotic events from single synapses or varicosities and providing statistically signicant quantitative and kinetic information achievable by no other methods.…”