Probing Respiratory Chain Function of Isolated Mitochondria Using Scanning Electrochemical Microscopy

Mitochondria are small organelles found within cells and are responsible for producing energy during respiration. Respiration is the process by which electrons from the oxidation of glucose are passed between enzymes located in the inner membrane of the mitochondria until they are used to reduce oxygen to water. During this process H+ is pumped across the membrane from the inside of the mitochondria to the outside, to produce a gradient in proton concentration. This proton gradient is the driving force for the production of ATP - the molecule that provides the energy for most cellular processes. It is interesting to study the process of respiration in mitochondria as it is believed that damage to the enzymes involved leads to the production of free radical oxygen species, like superoxide. Superoxide molecules have an unpaired electron and so are very reactive and immediately will react with surrounding molecules like DNA causing damage within the cell. These processes are implicated in several debilitating diseases such as Parkinson's and Alzheimer's as well as in aging processes and the mechanisms by which our cells die. This project studies these processes using a technique called Scanning Electrochemical Microscopy (SECM). SECM uses a very small electrode of diameter about one hundredth of a millimeter located very closely to the surface of a mitochondrion. The mitochondria have previously been isolated from cells and immobilised onto the bottom of a glass dish. The electrode can detect different molecules and measure the rate at which they are produced by the mitochondria. If we add chemical species that can be reduced by the respiratory enzymes, then the electrode can detect how fast they are reduced and how well the mitochondria are respiring. We can also deliberately add chemicals that damage the enzymes at defined places and measure how this changes the respiration of the mitochondria. In other experiments we can design special electrodes that detect superoxide radicals only and use them to determine under which conditions superoxide radicals are produced. This may help us to understand how damage to the respiratory chain enzymes in mitochondria leads to health problems in humans.