Mitochondria-Targeted Ratiometric Fluorescent Nanosensor for Simultaneous Biosensing and Imaging of O₂^•– and pH in Live Cells

Abstract

Intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis. It is also involved in various metabolic processes occurring in mitochondria, such as the generation of reactive oxygen species, calcium regulation, as well as the triggering of cell proliferation and apoptosis. Meanwhile, the aberrant accumulation of O within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development. To disentangle the complicated inter-relationship between pH and O in the signal transduction and homeostasis in mitochondria, herein we developed a mitochondria-targeted single fluorescent probe for simultaneous sensing and imaging of pH and O in mitochondria. CdSe/ZnS quantum dots encapsulated in silica shell was designed as an inner reference element for providing a built-in correction, as well as employed as a carrier to assemble the responsive elements for O and pH, together with mitochondria-targeted molecule. The developed nanosensor demonstrated high accuracy and selectivity for pH and O sensing, against other ROS, metal ions, and amino acids. The remarkable analytical performance of the present nanosensor, as well as good biocompatibility, established an accurate and selective approach for real-time imaging and biosensing of O and pH in mitochondria of live cells.

Development of a Modular Ratiometric Fluorescent Probe for the Detection of Extracellular Superoxide

“…Indeed, as also reported by two other groups that modified hydrocyanines for imaging purposes (derivatizing HCy5 with PEG or coupling them to a nano‐carrier), the detected response towards superoxide and hydroxyl radicals was lower compared to the original report . All these findings suggest that coupling the (hydro)cyanines to a macromolecule may influence the fluorescence properties and/or sensing ability and may account for the reduced sensing capacity and a high LOD (3.5 and 15.3 μ m ) compared to other examples in literature (80–90 n m ) . However, cyanines are also known to form H‐aggregates in water and buffer especially at higher concentrations, which may also occur with hydrocyanines and alter their sensing behavior .…”

“…Although selective superoxide detection has been improved recently, the sensing unit used in ratiometric probe designs is mostly hydroethidin (HE) . The reliability of this fluorophore indeed is plagued by several side reactions, for example, with cytochrome c .…”

“…[18][19][20] Although selectives uperoxide detection has been improved recently, [13,[21][22][23] the sensing unit used in ratiometric probe designs is mostly hydroethidin (HE). [24][25][26][27] The reliability of this fluorophore indeed is plagued by several sider eactions, for example,w ith cytochrome c. [28] Additionally,t hese probesd etect intracellular superoxide and, to the best of our knowledge, extracellulardetection of O 2 C À has not been attempted with ratiometric fluorescent probes. Consideringt hat the most specific and used technique is still the colorimetricc ytochromec reduction, [28] af luorescent tool to study extracellulars uperoxide specifically can be of great value.…”

“…[41,42] All these findings suggest that coupling the (hydro)cyanines to am acromoleculem ay influence the fluorescence properties and/or sensing ability and may account for the reduced sensing capacity and ah igh LOD (3.5 and 15.3 mm)c ompared to other examples in literature (80-90 nm). [26,27,43,23] However,c yanines are also known to form H-aggregates in water and buffer especially at higherc oncentrations, which may also occur with hydrocyanines and alter their sensing behavior. [44] What is worth noting is that, in the cell assays,t he increase in the fluorescent signal obtained with our ratiometric probe was in the same order of magnitude as that reported with the commercially availableH Cy5 (ROSStar 650).…”

Pseudomonas Quinolone Signal molecule PQS behaves like a B Class inhibitor at the I Q site of mitochondrial complex I

“…In HeLa cells, the effect of Rotenone was different: Rotenone significantly increased superoxide levels. It has to be mentioned that the effect of Rotenone on ROS production in cells is not consistent, depending on the ROS defense, which is obviously specific in A594 cells . To investigate whether Rotenone would in principle increase ROS in A594 cells, we additionally performed inhibitor treatment with isolated mitochondria from A549 cells based on observations that the ROS defense mechanism is often lost during mitochondrial isolation .…”