New Strategies Of Intracellular Component-Assisted Fluorescence Amplification

Intracellular biochemical information by using in-situ real-time monitoring technique as the cornerstone for exploring the nature of life phenomena,which is one of the core contents of current chemical biology research.With the abilities of high spatial and temporal resolution,non-destructive analysis,and easy operation,fluorescence sensing and imaging technique provide an indispensable research method for in-situ detection of intracellular interesting targets and their biological processes.However,current fluorescent biosensing techniques are difficult to in-situ monitor intracellular some low-abundance vital molecules and major diseases related markers.The key to real-time monitoring of low-abundance intracellular targets is to develop a versatile and biocompatible fluorescence amplification method,which could break through the one-to-one signal response mode of traditional fluorescent probes to solve the problem of existing fluorescence amplification by using foreign materials and harsh reaction conditions.It is also a major scientific problem that needs to be solved in the field of biomedical analysis.Based on the current problems in biomedical and analytical sensing,we are focusing on the development of a new method of intracellular in-situ fluorescence amplification.According to the biological characteristics of the interesting targets,we had proposed a kind of intracellular components to assist fluorescence amplification by using intracellular intrinsic components as signal amplification aids,which provide new ideas for the development of high-efficiency in vivo fluorescence imaging probes to explore the relationship between the change of intracellular bioactive substances and vital movement and the key markers of major diseases.The main works are listed as follows:（Ⅰ）Investigation on RNA-binding fluorescence amplification for the detection of intracellular specific biomolecules.（1）By using the fluorescent dye RBF with RNA-binding signal enhancing ability,RNA-binding fluorescence amplification as a novel signal amplification strategy has been developed in this chapter.In addition,Hyal-1,which is closely related to tumors,has better degradation properties of hyaluronic acid（HA）than other hyaluronidase subtypes.Herein,we developed a fluorescence-amplified nanosensor RBF@CHA self-assembled from a kind of cholesterolamine modified HA and the fluorescent dye RBF for intracellular Hyal-1 specific detection and imaging analysis.The experimental results showed that the nanosensor could not only achieve specific response to Hyal-1,but also exhibit the unique high sensitivity characteristics owing to the RNA-binding fluorescence amplification.Therefore,the nanosensor successfully achieved in situ imaging of intracellular low-abundance bioactive molecules.（Chapter 2）.（2）To further validate the universality of RNA-binding fluorescence amplification in biosensing applications.Based on this strategy,we selected cobalt hydroxide（CoOOH）with fluorescence quenching property as the responding device to encapsulate RBF in mesoporous silicon nanoparticles（MSN）to obtain the nanosensor for specific detection of intracellular ascorbic acid（AA）.The experimental results showed that the nanosensor could successful detection of intracellular AA with high sensitivity,and further verify the RNA-binding fluorescence amplification strategy with great potentials for intracellular in-situ detection（Chapter 3）.（Ⅱ）Investigation on GSH-assisted fluorescence amplification for the specific detection of intracellular oxygen content.The detection of intracellular oxygen content is of great significance for the study of its physiological and pathological mechanisms,but it still lacks a kind of oxygen sensors specifically for intracellular oxygen content detection.Herein,we had proposed a reduced glutathione（GSH）fluorescence amplification is used to construct intracellular oxygen sensors.In this chapter,we choosed the complex ofβ-cyclodextrin（β-CD）and low-oxygen-sensitive azobenzene as the gate device to encapsulate the GSH-sensitive fluorescent dye N-bMBN in MSN to obtain an intracellular oxygen sensor icO₂-GASensor.The experimental results showed that the nanosensor not only successfully used in specific detection of intracellular oxygen content due to the fluorescent dye N-bMBN with the ability of responding for GSH,but also achieved signal amplification with the help of MSN（Chapter 4）.（Ⅲ）Investigation on viscosity-assisted fluorescence amplification in-situ imaging of lysosomal cathepsin B.Lysosomal proteses have closely relationship between with the physiological and pathological functions of cells,but it is still very challenging to achieve in-situ high sensitivity detection of lysosomal protease activity in vivo.To solve this problem,we had proposed a kind of lysosomal viscosity-assisted fluorescence amplification strategy for the in-situ detection of lyasosomal cathepsin B（Cath B）.In this chapter,a Cath B-specific cleavage peptide was choosed to encapsulate the viscosity-sensitive fluorescent dye HPI inside the MSN,and then we could obtain a fluorescence amplification nanosensor Lyso-FA.The experimental results showed that the nanosensor could be successfully enter the intracellular lysosomes via endocytosis pathway,and achieve high sensitivity detection and in-situ imaging of Cath B at the subcellular level（Chapter 5）.The intracellular component-assisted fluorescence amplification proposed in this paper not only provides a kind of new ideas for signal amplification technology,but also promotes the development of relevant in-depth research in the field of biomedicine.Furthermore,this strategy provides basic theoretical guidance and large biochemical analysis application value for the development of in-situ hypersensitive visual analysis in living cells.