New Strategies Of Biological Sensing For Fluorescence Imaging

The development of new technology of biological fluorescence imaging is the hot frontier in the field of analytical chemistry and life sciences.Developing new biosensing technology with high sensitivity and selectivity plays an important role for imaging and analyzing tumor cells and important signal molecules,proteins and ions in the cell.It is well known that nucleic acids not only play an important role in organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins,but also have high specificity and affinity for different targets including nucleic acids,proteins,small molecules and ions,and are therefore establishing nucleic acid-based bioluminescent probes has attracted a great attention of many researchers.Biosensors combined with nanotechnology effectively improves the sensitivity,stability and multifunctional properties due to their nanosized structure,large surface area,high catalytic efficiency and good biocompatibility,which also provide novel strategies for the development of biological fluorescent probe.In addition,the use of nanotechnology for early diagnosis and treatment of cancer are proceeding all over the world,nanomedicine and gene therapy have been achieved great progress in clinical studies.Nanotherapy will be a more effective strategy for the treatment of cancer among radiotherapy,chemotherapy and surgery.This doctoral thesis developed a series of new biosensing strategy by coupling nucleic acid probe,polymer nanomaterials and organic small molecular fluorescent probe for visualization of KRAS point mutation and microRNA expression in situ,bioimaging of intracellular lysosomal pH changes and hypochlorous acid in living cells,as well as the use of high biocompatibility peptide nanomaterials for targeted therapy and fluorescence imaging of tumor cells.The detailed contents are described as follows:Development of biocompatible chemical tools for precise cancer theranostics and imaging remains a priority in chemistry and biomedicine.In Chapter 2,we develop a novel activatable cancer theranostic probe using highly biocompatible self-assembled peptide nanowires which are activated by tumor-associated peptidases to realize targeted therapy and imaging.We designed and synthesized a four-domain peptide containing in tandem a self-assembling peptide domain Q11,a cytotoxic peptide melittin,a peptidase substrate and a polyanionic sequence.The nanowires are synthesized readily using the fibrillizing peptide domain via mix assembly with a fluorophore labled Q11 peptide and display multiple activatable cytotoxic peptides on the surface.The surface-displayed peptides can be cleaved by matrix metalloproteinase 2(MMP2),releasing the toxicity-neutralizing domains,which effectively activates the cytotoxic peptides for targeted theranostics and fluorescence imaging.The developed activatable probe holds great potential for sensitive diagnosis and efficient therapy of cancer.Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics.In Chapter 3,we develop a novel fluorescence in situ hybridization(FISH)strategy based on a new branched hybridization chain reaction(bHCR)for efficient signal amplification in FISH assay and a ligase-mediated discrimination for specific mutation detection.To our knowledge,this is the first time that HCR has been realized for mutation detection in FISH assay.In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA,and generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR.Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules,and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA.Moreover,this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single cell level.Therefore,this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications.MicroRNAs(miRNAs)are small regulatory RNA,which have a variety of biological funtions and are closely related to various diseases.Quantification and visualization of the spatial distribution and expression levels of miRNA in situ at single-cell level is very important for study miRNA-mediated regulatory netmorks and coplex biological functions.In Chapter 4,we use the branched hybridization chain reaction(bHCR)for quantitative imaging of the expression of miRNA at the single cell level in cancer cells.In order to reduce the loss of miRNA by traditional paraformaldehyde immobilization,miRNA were covalently cross-linked to the protein by EDC and paraformaldehyde,and the miRNA expression level was detected by bHCR using the target miRNA as the initiation strand.The imaging results show that this stratege provide highly sensitive and selective for visualization of miRNA at single cell level,as well as differentiate miRNA expression levels in different cells.Comparing with FISH,we propose that target-based bHCR-FISH in situ assays provide a reliable analytical platform for the study of disease related with miRNA expression profiling.Intracellular pH balance plays a key role in cell behavior and pathology,the design of effective tools capable of sensing lysosome pH is highly desirable for better understanding its biological functions in cellular behaviors and various diseases.In Chapter 5,a lysosome-targetable ratiometric fluorescent polymer nanoparticle pH sensor(RFPNS)was synthesized via incorporation of miniemulsion polymerization and surface modification technique.In this system,the donor:4-ethoxy-9-allyl-1,8-naphthalimide(EANI)and the acceptor:fluorescein isothiocyanate(FITC)were covalently linked to the polymer nanoparticle to construct pH-responsive fluorescence resonance energy transfer(FRET)system.The FITC moieties on the surface of RFPNS underwent structural and spectral transformation as the presence of pH changes,resulting in ratiometric fluorescent sensing of pH.The as-prepared RFPNS displayed favorable water dispersibility,good pH-induced spectral reversibility and so on.Following the living cell uptake,the as-prepared RFPNS with good cell-membrane permeability can mainly stain in the lysosomes;and it can facilitate visualization of the intracellular lysosomal pH changes.This nanosensor platform offers a novel method for future development of ratiometric fluorescent probes for targeting other analytes,like ions,metabolites,and other biomolecules in biosamples.Hypochlorous acid(HOC1),one of those important reactive oxygen species(ROS)in living organisms.Endogenous HOC1 is mainly produced by myeloperoxidase-cataly zed peroxidation of chloride ions in white blood cells,which are closely linked to the immune defense system.In chapter 6,a novel activatable fluorescent probe for hypochlorous acid(HOC1)imaging has been developed based on HOCl-triggered aldehyde recovery reaction.This probe features with a long emission wavelength because of the use of intramolecular charge transfer(ICT)effect in the fluorophore.Moreover,this probe displayed ultrafast reponse(within Is),good selective and high sensitivity(detection limit of 50nm)toward HOC1 with excellent independency upon pH of the assay system.Live cells imaging studies demonstrate that the probe can be applied successfully to detect exogenous and endogenous HOC1.