Design Of Monomer/Excimer Conversion-based Fluorescent Sensors And Their Applications In Fluorescent Detection

Dithiothreitol is a potent reducing agent for protecting sulfhydryl groups and has been widely applied in clinical and laboratorial fields of biochemistry, cell biology and pharmacy. The abnormal level of dithiothreitol may damage redox balance of peptide, protein and DNA, resulting in destruction and dysfunction of some biomacromolecules in the experiments. The ubiquitous use of dithiothreitol in biology, biochemistry and ecology makes it a challenge for discriminating dithiothreitol from biothiols. For some precision instruments or therapeutic systems, temperature and its distribution are required to be kept monitoring, so as to prevent any accidents. Few of conventional thermometers can accurately measure temperature with high spatial resolution under complicated circumstance, so it is meaningful to find novel thermometers which can work in complex environment.Fluorescent sensors exhibit high sensitivity, good selectivity, non-destruction and operation convenience, maintaining good momentum of development in many fields. Moreover, there are many kinds of detection mechanisms for fluorescent sensors, which help to overcome some common detection problems. In this paper, we select pyrene as the fluorophore, which possesses high quantum yield and monomer/excimer fluorescence interconversion, and design two fluorescent detection systems respectively. The results are illustrated as follows:(1) We have prepared a fluorescence turn-on sensor by linking a pyrene fluorophore to a maleimide group, for maleimide group is highly responsive to sulfydryl-containing compounds through Michael addition; and it is also capable of quenching the emission of pyrene through photoinduced electron transfer process. By utilizing the monomer/excimer conversion of fluorescence emission, it can discriminately and simultaneously detect dithiothreitol and single sulfydryl-containing thiols. The structure of fluorescent sensor was characterized by mass spectra, 1H-NMR and 13C-NMR, and the detection mechanism was validated by mass spectra and high performance liquid chromatography respectively. This good selective sensor is able to quantitatively detect dithiothreitol in low concentration range and the detection limit is determined to be 0.07 μM. Furthermore, the probe can be applied to fluorescence imaging in cell and detecting dithiothreitol in biological milieu.(2) We synthesized positively charged pyrene derivative(Py-N) and combined it with negatively charged poly(sodium 4-styrenesulfonate)(PSS) in PBS buffer to form a thermo-sensitive fluorescent system. Taking full advantage of polyelectrolyte‘s electrostatic interaction and multiple anchoring sites along macromolecular chain could greatly facilitate the excimer/monomer conversion of Py-N caused by temperature variation in the system. The structure of Py-N was characterized by mass spectra and 1H-NMR, and a suitable ratio of Py-N and PSS was determined by experiment in order to ensure optimal performance of the thermo-sensitive fluorescent system. The thermo-sensitive fluorescent system exhibits quite good thermo-sensitive feature and thermo-responsive reversibility. The fluorescence intensity ratio versus temperature curve for the system can be fitted by a fifth-order polynomial(R2=0.9997). By using CIE diagram to describe fluorescence signal changes resulted from temperature variations, we can draw a conclusion that this thermo-sensitive fluorescent system shows great potential for serving as a fluorescent thermometer in complex environment.