Design And Synthesis Of A Novel Chemiluminescence/fluorescence Dual Probe For The Detection In Bioactive Molecule

As we know, aerobic organisms can’t live without oxygen, including humans.Aerobic respiration process produces a variety of reactive oxygen species, such asreactive oxygen species (including O2·-,·OH,1O2, H2O2, HClO, etc), reactive nitrogenspecies (including ON and ONOO-, etc) and reactive sulfer species(including H2S,etc).These reactive species are closely implicated in a wide range of physiological andpathological processes, such as a variety of biochemical reactions, the regulation ofapoptosis and proliferation, cell signal transduction, etc. Although they are the necessarymetabolites of the cell, when overproduced, they can also cause various diseases throughthe oxidation of biomolecules, such as lipids, proteins and DNA.Although the content of these active species is not high, they play an important roleon the health of the human boy. So to clarify their specific physiological and pathologicalmechanism has become an important research field. As we know, these active speciesshow characteristics in terms of short life time and high oxidation activity. What’s more,as there are many antioxidants in organism, it is difficult to detect the accurate content ofthese active species. One of the difficulties of this stage study is the lack of suitabledetection methods. To date, several methods for active species detection include electronspin resonance spectroscopy (ESR), high performance liquid chromatography (HPLC),chemiluminescence, electrochemical methods and fluorescence, etc. Fluorescencedetection is highly sensitive and selective, as well as relatively simple. Combined withlaser scanning confocal microscopy,“real-time, visualizing, quantitative and no damage”detection of reactive oxygen species can be achieved. However, this method is susceptibleto the autofluorescence interference by organisms because of its short excitationwavelength. Chemiluminescent has fast reactive speed and does not need excitation light souce. It can realize the instantaneous detection of the reactive species with highsensitivity.In this paper, we carried out the research work as follows by combining theadvantages of fluorescence and chemiluminescence method.First, the design and synthesis of a novel fluorescence/chemiluminescence dualprobe (Indole-CLA) for the detection and imaging of O2·-in living cells and animals wasaccomplished. Indole-CLA was synthesized by linking superoxide anion-specific groupsCLA and fluorophores of merocyanine, and was proved to be a dual-modalfluorescence/chemiluminescence probe for superoxide with turn-on response. Thefluorescence mode (λex/λem=480/554nm) for superoxide holds a standard equation ofY=3411.7+5122.5[O2·-](μM) and a detection limit of4.03nM, respectively. The standardequation of the chemiluminescence mode (luminescence at554nm) holds a standerdequation of Y=11.60+263.90[O2·-](μM) and a detection limit of26.2nM, respectively.Using laser scanning confocal microscopy, the superoxide anion radical that produced inthe PMA-stimulated murine macrophages (RAW264.7) was analyzed successfully byfluorescence method. Using SynergyMx Multi-Mode Microplate Reader(BioTekInstrument, Winooski, VT, USA), the superoxide anion radical that produced in thePMA-stimulated murine macrophages (RAW264.7) was analyzed successfully bychemiluminescence method. Using UVP ibox600animal imaging system, thesuperoxide anion radical that produced in the LPS-stimulated BALB/c mice was analyzedsuccessfully. The results provided a new fluorescence/chemiluminescence dual-modalprobe for superoxide anion radical in living cells and animals, whice show goodapplication prospect.