Nucleic Acids Assay Based On Soluble Carbon Nanoparticles

Most of life processes are closely associated with nucleotic acids. So detection of nucleicacids and their mutation (especially for single-nucleotide polymorphism,SNP) plays importantroles in life science research,development of new kinds of drugs,and early disease diagnosis.Developing simple,rapid and specific methods for detecting nucleic acids and SNP is ofgreatly significant. This dissertation focuses on a new soluble carbon nanoparticlespreparation (CNPs),characterizations and development of CNPs-based methods for detectionof nucleic acids and SNP in homogeneous solution. The main contents are as follows:1. CNPs with perfect water solubility and biocompatibility,narrow particledistribution,high fluorescence quenching effiency were prepared by simple electrochemistrymethod. The mophyologies of CNPs were characterized by Transmission ElectronMicroscopy (TEM),Scanning Electron Microscopy (SEM) and Atomic Force Microscopy(AFM),and the particle size distribution was measured by laser particle analyzer. Thestructure of CNPs was also characterized with X-Ray powder diffraction system,andinformations of functional groups on CNPs were idenrified by infrared spectrum. Themechanism of quenching fluorescence by CNPs was studied by using soluble conjugatedfluorescent polymer (PFP).2. Based on the different interactions between sp2hybrid carbon atoms and singlestranded or double stranded nucleotic acid,a simple and fast detection method for DNA andSNP was developed. The DNA in the concentration range of 1~200 nmol/L could bedetectable,and the detection limits were respectively 1.7 and 0.8 nmol/L with differentfluorescent molecules labeled on DNA probes. Furthermore,a fast and accurate assay of SNPand mismatch bases in DNA was developed based on differences of denatured temperaturesbetween perfect match double stranded DNA and the double stranded DNA with mismatchbases.3. Based on the high efficiency of fluorescence quenching and the different affinities ofwater-soluble CNPs towards single-stranded DNA (ssDNA) and double-stranded DNA-RNA hybrid, a novel, rapid and cost-effective assay for detection of miRNA and nuclease activitywas developed. The fluorescein-labeled ssDNA probe (FAM-P) could be absorbed on thesurface of CNPs throughπ-πstacking interaction giving rise to fluorescence quenching. Byintroduction of microRNA complementary to the DNA probe, the double-stranded DNA-RNAhybrid could be formed and released from the surface of CNPs resulting in the fluorescencerecovery. Thus, miRNA was successfully detected in homogenous fashion without anyamplification or enzyme involving reactions. Moreover, we demonstrated that nucleaseactivities of RNase H and DNase I could also be sensitively monitored by using CNPs basedon the fluorescence changing of DNA probe. So, the CNPs provide an excellent homogeneoussensing platform for studying on the molecular diagnosis and therapeutics.