Nucleic Acid Probe-based Fluorescent Biosensor For Intracellular MRNA And PH Imaging

The development of life science and technology is closely related to the diagnosis and treatment of disease,food safety and environment sanitation in life.How to use life science technology to improve “quality of life” is one of the urgent problems to be solved in global research work.Biosensor technology is active multi-disciplinary analytical technology that can be used to continuously,quickly and sensitively monitor key parameters in these fields.They have been given unique optical properti es and mechanical stability due to the continuous development of nanomaterials and technologies,which has prompted researchers to explore it more deeply at the molecular level.In recent years,the combination of biosensors with nucleic acid signal amplification and DNA nanostructure self-assembly technology has provided a powerful strategy for intracellular detection and promoted the development of biosensor in complex biological environments.In this paper,a new fluorescence biosensor method for detection of tumor-associated mRNA and pH was established by combining isothermal non-enzymatic nucleic acid signal amplification technique and DNA nanostructure self-assembly method,achieving detection and imaging of target analytes in living cells.The specific contents of the study are as follows: 1.Catalytic Hairpin Assembly-Based DNA Probe for Tumor-related mRNA Imaging in Living CellsMessenger RNA(mRNA)is a low abundance biomarker of early diagnosis of diseases,which plays an important role in the process of life.The expression level of mRNA can provide valuable information for medical diagnosis.Therefore,it is very essential to develop a rapid and accurate strategy for detection and analysis of mRNA in living cells.In chapter 2,we develop a signal amplification and diagnosis system,which uses catalytic hairpin assembly(CHA)technology to enhance fluorescence signal for imaging of tumor-related mRNA in living cells.CHA-based biosensor mainly depends on the hairpin structure of H1 and H2.The complem entary bases sequences of H1 and mRNA were modified by fluorophore and quencher.In the absence of tumor-related mRNA in the system,the hairpin structure of H1 and H2 remains stable in the solution due to hybridization of their loopbase sequences,and the fluorescence is effectively quenched.Once the target mRNA appeared in the system,CHA amplification reaction could be initiated via hybridization assembly of two hairpins,resulting in fluorescence recovery.When two hairpins are into living cells,the low abundance of mRNA activates CHA,and then produce multiple dsDNA products to achieve sensitively mRNA imaging in living cells in response to a single mRNA.Therefore,this biosensor based on CHA signal amplification can be used as an effective tool for early diagnosis of disease.2.DNA Nanoprobe for Imaging in Living CellspH is an important physiological parameter in living organisms,related closely to various cellular events and life activities.A slight change in pH can have a great impact on various metabolic activities in the process of life,not only breaking the balance of homeostasis,but also causing various physiological disorders.Therefore,in Chapter 3,we propose a reversible and stimulus-responsive DNA nanoprobe for intracellular pH imaging.The DNA nanoprobe biosensor is composed of two assembly module of Y-shaped dsDNA(Ya and Yb)and one linker module of linear dsDNA(linker).Among them,the linker contains a quencher-labeled i-motif DNA strand and a complementary strand labeled with a fluorophore.Under weak acid condition,the i-motif strand is forced to fold into a C quadruplex to separate from DNA nanostructure skeleton,and then fluorescence is restoredin a "signal-on" state;when the system is in a weakly alkaline condition,linker doesn't change its conformation and maintains rigid dsDNA helix,so DNA nanostructure is complete.Due to the close proximity of fluorophore and quencher,the fluorescence is effectively quenched,and DNA nanoprobe can quickly respond to pH fluctuations.This design confers a high sensitive local fluorescence of DNA nanoprobe,and DNA nanoprobe exhibit a reversible dynamic response in the physiological pH range,demonstrating their potential in in vitro pH detection and imaging in living cells.In additi on,this design can also connect different functional elements into the assembly modules of DNA nanostructure for other biomolecules detection.Therefore,the DNA nanoprobe-based biosensor platform have great potential in pH-related disease diagnosis,drug controlled release systems and other fields.