Design And Application Of Functional Fluorescent Probe In Bioanalysis

Fluorescence analysis is the most commonly used method for biological detection.It has the advantages of good selectivity,sensitivity,accuracy,speed,convenience,simplicity and wide linear range.Therefore,it is widely used in life analysis,medical testing and environmental monitoring.Signal pathways and signaling molecules play a vital role in life analysis,and fluorescent probes are the most powerful tools for studying signaling pathways and signaling molecules.With the deepening of research on life analysis,the demand for fluorescent probes is getting higher and higher,so we need to develop more new ideas to design fluorescent probes.Based on this,this paper is dedicated to the design of novel functionalized fluorescent probes and their application to cell biosensing analysis.The main research contents are as follows:1.A fluorescent lifetime probe:quantitative imaging of ultra-trace endogenous hydrogen polysulfide in cells and in vivoHydrogen sulfide(H2S)has been recognized as an important endogenous gasotransmitter associated with biological signaling transduction.However,recent biological studies implied that the H2S-related cellular signaling might actually be mediated by hydrogen polysulfides(H2Sn,n>1),not H2S itself.Unraveling such a mystery strongly demanded the quantification of endogenous H2Sn in living systems.However,endogenous H2Sn has been undetectable thus far,due to its extremely low concentration within cells.Herein,we demonstrated a strategy to detect ultra-trace endogenous H2Sn via a fluorescent lifetime probe,through changes of fluorescence lifetime instead of fluorescence intensity.The probe consists of a recognition group that specifically responds to H2Sn and a fluorescent group fluorescein.This fluorescent lifetime probe exhibited an ultrasensitive response to H2Sn,bringing about the lowest value of the detection limit(2 nM)and a lower limit of quantification(10 nM)to date.With such merits,we quantified and mapped endogenous H2Sn within cells and zebrafish.The quantitative information about endogenous H2Sn in cells and in vivo may have a significant implication for future research on the role of H2Sn in biology.The methodology of the fluorescence lifetime probe established here might provide a general insight into the design and application of any fluorescent probes,beyond the limit of utilizing fluorescence intensity.2.Cascade energy transfer fluorescent probe for real-time monitoring of drug delivery in living cellsIt is a great challenge to monitor drug release in live cells and design a drug delivery system with a controlled manner,especially one triggered by an exclusive endogenous disease marker and with an easily tracked release process.Herein,we developed a drug delivery platform of carbon dots which were connected to a stem-loop molecular beacon loaded with doxorubicin and polyethylene glycol modified folic acid.Such a platform enables one to release drugs on demand under the stimuli of endogenous microRNA-21,and turn on the fluorescence of carbon dots and doxorubicin,which allows one to monitor the drug release process.The intracellular experiment indicated that folic acid could mediate endocytosis of the nanocarrier,and the overexpressed endogenous microRNA-21 served as a unique key to unlock the drug nanocarrier by competitive hybridization with the molecular beacon,which finally resulted in fluorescence recovery and realized a chemotherapeutic effect within human breast cancer cells.The nanocarrier may have potential application in personalized treatment of different cancer subtypes in which the corresponding miRNAs are overexpressed.3.Kinetics study of single molecule by plasmon-modulated fluorescent probe Molecular beacons are a class of DNA strands that have been specifically designed to have a special sequence hairpin structure,and their specific recognition ability is widely used in fluorescent probes.Molecular beacons can be applied to genetic screening,biosensor development,biochip construction,detection of single nucleotide polymorphisms,and mRNA monitoring in living cells.The signal transduction mechanism of the molecular beacon enables it to analyze the target oligonucleotide without futher separation of the unbound probe.The stem-loop structure allows the fluorophore and the quenching group to be in close proximity to each other,which resulted in the quenching of fluorescence.When hybridization occurs,the molecular beacon spontaneously undergoes a conformational change,thereby separating the fluorophore group and the quenching group while restoring fluorescence.The separation of the fluorophore group and the quenching group is a critical step in the generation of changes in the fluorescent signal.Since this process is rapid,it is difficult to monitor the effects of various factors in the environment on the process.Here,we constructed a new molecular beacon model by linking gold nanoparticles and CdS quantum dots through molecular beacon.We used dark field microscopy transmission grating to observe the change of the spectrum peak position of the probe,which successfully monitored the environmental impact of a single molecule on a single particle when open the molecular beacon.At the same time,the effects of interference factors such as photobleaching were avoided.