A new ratio fluorescence probe: it can locate mitochondria and respond to calcium ions

Ca2 + is an important metal ion which is essential for physiological activities of living body Its concentration and distribution are very important for maintaining the biological potential on both sides of cell membrane, normal nerve conduction function, normal muscle expansion and relaxation function and the regulation of some hormones As an important intracellular calcium bank, the content of calcium in mitochondria is nearly one thousand times higher than that in cytoplasm, and it has a perfect Ca 2 + uptake and release system In addition, mitochondria are the center of cell energy metabolism The Ca2 + in mitochondria is involved in the pathological process of many diseases, such as ischemic brain injury, Alzheimer's disease, Parkinson's disease, type II diabetes, epilepsy, etc Therefore, the measurement of the concentration and distribution of Ca2 + in mitochondria at the subcellular level can not only help us better understand the Ca2 in the organism+ Signal transduction can also help researchers systematically understand the role of Ca2 + in physiological and pathological processes Figure 1 Synthesis path of calcium ion probe (image source: angelw Chem Int ed.) recently, Dr Diana pendin and Dr Andrea mattarei from the University of Padua, Italy, jointly reported a fluorescent probe that can be located in mitochondria The UV absorption, fluorescence excitation and emission patterns of the probe will change obviously before and after the combination of calcium ions Based on this characteristic, the author used the probe as a calcium ion probe to image the distribution of calcium ions in mitochondria of living cells This achievement was published in German Applied Chemistry (DOI: 10.1002 / anie 201902272) under the title of "asynchronous fluorescent mitochondria targeted sensor for rapid imaging of chemistry in live cells" The synthesis of Ca2 + probe can be divided into four steps: recognition of Ca2 + parent synthesis, modification of fluorophore, modification of localization group and modification of cell penetrating auxiliary group am Firstly, the derivative A10 of BAPTA with calcium ion chelating structure was prepared by using hydroquinone as raw material through 10 steps reaction, which introduced 4-chlorobutyl to react with triphenylphosphine on the basis of BAPTA; then, the fluorescent group was introduced into the structure of A10 through three steps reaction and the alkyl chloride for reaction of locating group was connected at the end of the fluorescent group Then, the probe intermediate A15 containing two triphenylphosphines was obtained by the reaction of halogen and triphenylphosphine Finally, the probe mt-fura-2-am containing am group was obtained by hydrolysis and addition Fig 2 Location of mt-fura-2-am and its fluorescence imaging of calcium ions (image source: angel Chem Int ed.) Because the probe contains a structure that can bind calcium ions, the author first studied the change of its spectral properties through ultraviolet and fluorescence The results show that the maximum absorption wavelength and emission wavelength of the probe before and after binding calcium ions are 385 respectively The blue shifts of nm and 540 nm to 345 nm and 520 nm, while the maximum excitation wavelength shifts from 350 nm to 400 nm In addition, the co localization experiment with mitochondrial fluorescent protein showed that the synthesized probe could indeed enrich into mitochondria after entering cells as expected Based on these results, the response of the probe to the change of calcium concentration in cells was studied The results show that under 350 nm excitation, the fluorescence signal increases slightly after combining with calcium ion, while under 400 nm excitation, the fluorescence signal decreases significantly, and the ratio of the two changes as high as 300% before and after combining It is worth noting that compared with the previously reported mitochondrial calcium probe based on Rhodamine, the newly prepared mt-fura-2-am has higher photostability and will not easily leak out of the mitochondria In the end, the real-time analysis of calcium flow in HeLa cells was carried out by using the probe, and the growth of calcium ion in HeLa cells stimulated by KCl was successfully observed Full text authors: Diana pendin, Rosa norante, Andrea de Nadai, Gaia Gherardi, Nicola vajente, Amy Basso, Nina kaludercic, Cristina mamucari, Cristina paradisi, Tullio pozzan and Andrea mattarei.