Construction And Sensing Performance Of A Novel Ratio Fluorescent Probe

With the rapid development of life sciences,small molecules,nucleic acids,proteins and other large molecules in the organism participate in metabolism and maintain their life activities,so the detection of biomolecules is extremely important.Currently,in the fields of clinical diagnosis,environmental monitoring and biological analysis,there are many methods to detect biomolecules,such as chromatography,electrochemical method and fluorescence method,etc.,among which fluorescence method is favored by people owing to the advantages of simple operation,low cost and good repeatability.However,most fluorescent probes only have single detection response signals,which can easily affect the detection accuracy under the interference of external factors,such as experimental instruments,detection environment and other external factors.The ratio fluorescent probe(Ratio)is used the ratio of the intensity of two wavelengths as the output signal that has became a current research hotspot because of its high accuracy.Compared with enhanced(OFF-ON)or quenched(ON-OFF)fluorescent probes,ratio fluorescent probes have outstanding advantages in improving detection accuracy,such as providing internal correction and avoiding false positive signals.In recent years,ratio fluorescent probes have become an effective tool for real-time analysis and detection of biomolecules.In this paper,the ratio fluorescent probes of sulfur dioxide(SO₂),adenosine triphosphate(ATP)and micro RNA(mi RNA)were constructed based on the recognition mechanisms of intramolecular charge transfer(ICT)and fluorescence resonance energy transfer(FRET).The specific research contents are as follows:1.A BODIPY?Hemicyanine?Based Water?Soluble Dual?Color Fluorescence Probe for Ratio Monitoring of Intracellular Endogenous Sulfur Dioxide and Bioimaging ApplicationsAbnormal levels of endogenous SO₂ can cause toxic effects giving rise to cardiovascular disease,nervous system disorders,and cancer.Therefore,it is great significance and value to use effective molecular biology tools to monitor in detail the generation and physiological effects of endogenous SO₂.The dual-color fluorescence probes can provide the intensity change of two emission wavelengths when detecting the target,which are beneficial to realize the colorimetric detection,obtain more accurate results,and be more suitable for the monitoring applications in vivo.In recent years,many fluorescence probes have been reported for determination of SO₂ and its derivatives,and great progress has been made.However,most of the reported probes for determination of SO₂ have limitations.The biggest disadvantage is poor water solubility,which leads to dissatisfactory detection limit,long response time,and so on.Therefore,this limits their further application for the determination of intracellular endogenous SO₂ in biological environment.Herein,based on BODIPY and hemicyanine,we for the first time conjugated aldehyde-BODIPY with the water-soluble iodopropionic-acid-hemicyanine to develop a new dual-color fluorescence probe(BHC)for selective and sensitive monitoring of endogenous SO₂.Through excellent water solubility and the unique nucleophilic addition reaction,BHC has a higher selectivity for the colorimetric monitoring of SO₂ than other related substances.The resulting probe BHC has red emission,and can be converted to green emission after reacting with SO₂.Then the dual-color fluorescence probe was further applied to image intracellular endogenous generated SO₃^2-/HSO₃^-by the metabolism of living cells with satisfactory result.This strategy provides a new tool for revealing the metabolism of SO₃^2-/HSO₃^-and quantifying their distribution in living cells.2.A Ratio Fluorescence Nanoprobe Based on FRET for Monitoring Adenosine Triphosphate with High Sensitivity and SpecificityATP is the main energy source for life activities in organisms that plays a vital role in various life activities.In addition,ATP expression level is also related to many diseases.Therefore,the detection of ATP is of great significance for studying the cellular mechanism and clinical diagnosis.In this work,a fluorescent nanoprobe based on FRET was designed and synthesized to detect adenosine triphosphate(ATP).Synthetic fluorescent dye-doped silica nanoparticles(SNPs)were coated with carboxyl groups and covalently bonded to amino-modified hairpin primers through amide bonds,where the ends of the hairpin primers were modified with fluorescein(FAM).The ATP aptamer was hybridized with the hairpin primer,and the hairpin primer was opened to induce the formation of a fluorescent nanoprobe.In the presence of ATP,due to the high affinity between the target and the aptamer,the primer formed a hairpin again,causing FRET to occur between the two fluorophores of SNPs and FAM.The fluorescent probe had some excellent properties,such as rapid reaction,high sensitivity,and detection limit of ATP is0.067 m M.FRET-based strategy can avoid the false positive signal and eliminate the influence of the external environment.These results suggest that this novel fluorescent probe will provide a new detection platform for the study of many substances related to the activities of life.3.Ratio Fluorescent Nanoprobes Based on Signal Amplification Strategies and FRET for Micro RNA Detection with High Sensitivity and SelectivityThe expression level of mi RNA is closely related to many diseases and can be used as a tumor diagnostic marker.Detection of mi RNA has broad application prospects in the early diagnosis and treatment of tumors.Due to the small size and easy degradation of mi RNA,mi RNA detection methods need to have high sensitivity and accuracy.In this paper,fluorescent nanoprobes based on signal amplification strategy and FRET strategy were designed to detect mi RNA.The signal-amplifying nanoprobe(SANP)consisted of SNPs(silica nanoparticles as FRET donors),H1 and H2-FAM(FAM as FRET acceptors).First,H1 was fixed to SNPs through amide bonds to form SNPs-H1.When the target was present,the CHA process was triggered,and energy transfer occured between SNPs and FAM.The nanoprobe effectively eliminates the error caused by background interference through the fluorescence resonance energy transfer(FRET)mechanism.Importantly,this probe achieves enzyme-free cycling amplification of the target through catalytic hairpin assembly(CHA)with a detection limit of 33 p M,which has the advantages of high sensitivity and specificity.The detection of mi R-203 in vitro demonstrated the ability to monitor the expression levels of mi RNA,and these results indicate that the method provides potential applications for biomedical and clinical research on the expression level of biomolecules.