Design And Preparation Of Copolymer-based Fluorescence Sensor And Its Application For Homocysteine And Protein Phosphorylation

Fluorescent conjugated polymers,as a class of high molecular compounds with special optical and electrical properties,have received widespread attention in the fields of biosensors and cell imaging.The conjugated polymer has a?-?conjugated molecular wire structure,which can magnify the fluorescence response signal exponentially.This discovery provides a new way to develop highly sensitive fluorescent chemical sensors.Homocysteine?Hcy?level is one of the important indicators reflecting human health,and is closely related to the occurrence and development of cardiovascular and cerebrovascular diseases.It has been identified as an independent risk factor for diabetic nephropathy and cardiovascular and cerebrovascular diseases.Protein phosphorylation is an important protein post-translational modification process.Protein phosphorylation and dephosphorylation regulate protein function and are related to signal transduction.Abnormal protein phosphorylation is closely related to the occurrence and development of neurodegenerative diseases.Therefore,the development of fluorescence sensors for detecting Hcy and protein phosphorylation levels,and the rapid and sensitive detection and imaging analysis of Hcy and protein phosphorylation levels in living organisms have important practical significance for the early diagnosis and mechanism research of related diseases.Based on the above background,near-infrared conjugated polymer nano-fluorescent sensors for detecting Hcy and phosphorylation sites were designed and synthesized,respectively.The two types of sensors are use 2,7-dibromofluorene and 4,7-bis?2-bromothiophene?-2-1-3-benzothiadiazole as the main chain,and dipyridylamine?DPA?is attached to the main chain through six methylene side chains.The following research work has been carried out:1.Using 2,7-dibromofluorene and 4,7-bis?2-bromothiophene?-2-1-3-benzothiadiazole as copolymer fluorophores,Cu???chelated DPA was used as a recognition group to prepare a fluorescent nanosensor DPA-PFNP-Cu???that specifically recognized Hcy.Cu???is combined with the N atom in DPA through chelation to quench the fluorescence of DPA-PFNP-Cu???.Hcy and Cu???in the sensor DPA-PFNP-Cu???are used to compete for coordination to restore the fluorescence of the sensor.The spatial network structure enables specific detection of Hcy.DPA-PFNP-Cu???particle size and zeta potential were characterized by transmission electron microscopy and dynamic light scattering.The results showed that DPA-PFNP-Cu???particle size was 100 nm and had good stability.Selective experiments in vitro and in the cell environment have verified that DPA-PFNP-Cu???can selectively detect Hcy without being interfered by other thiol compounds.Finally,the sensor DPA-PFNP-Cu???was applied to a mouse model of diabetes to image Hcy levels in the kidney and liver,and found that Hcy levels were positively correlated with the degree of diabetes.More importantly,the fluorescence sensor DPA-PFNP-Cu???has deep tissue penetration ability,which makes it possible to detect Hcy in liver and kidney parts in a diabetic mouse model without damage.The experimental data show that the results of two-photon imaging and live imaging are consistent,and the mutual correction of the two methods improves the accuracy of the test results.2.On the basis of previous experiments,a fluorescent nanosensor DPA-PFNP-Zn???that specifically recognizes protein phosphorylation sites was prepared using a conjugated polymer of the above structure as a fluorophore and Zn???chelated DPA as a recognition group.The combination of Zn???and the N atom in DPA through chelation causes the fluorescence of the phosphorylation site sensor to be quenched.The competitive coordination of phosphorylation group and Zn???in the sensor DPA-PFNP-Zn???are used to restore the fluorescence of the sensor to achieve phosphorylation specific detection.The selectivity of DPA-PFNP-Zn???was characterized by fluorescence tests under different conditions,and the results showed that both dissociative PO₄^3-and phosphorylated proteins can cause an increase in fluorescence signals and unaffected by the widespread presence of phosphate ATP in organisms.This proves the feasibility of DPA-PFNP-Zn???as a fluorescent nanosensor for protein phosphorylation sites,and DPA-PFNP-Zn???can be applied to mouse models to achieve the detection of phosphorylation sites in vivo.