Graphene Oxide-based Fluorescent Nucleic Acid Biosensors

Molecular diagnostic techniques aiming at nucleic acid and proteindetection are playing an increasingly important role in the field of geneticdisease, tumor diagnosis, individualized health care, etc. Establishing ahighly sensitive, selective, simple and rapid biological detectiontechnology is of great significance for the development of moleculardiagnostics. In recent years, nanotechnology, as a rapid developmentinterdisciplinary, has become a hot research topic. Nano-biosensor is oneof the most attractive research directions in the field of nanotechnology,which has very important application prospect in biomedicine.A new kind of nanomaterial, graphene oxide (GO), it displays goodwater-solubility and flexible modification due to the massive suspendedhydroxyl and carboxyl groups present on the surface. In view of GO’sproperties of long-range nanoscale energy-transfer and adsorption withnucleobases or aromatic compounds noncovalently via π-stackinginteraction, two rapid, sensitive and selective nanobiosensors based onfluorescence for the detection of nucleic acids were developed in thisdissertation. The main contents are as follows:In Chapter1, we used the neutral PNA as a fluorescent nanoprobefor recognizing ssDNA with the platform of GO. First, the fluorescentdye-labeled PNA was physically absorbed on the surface of GO,accompanied with the quenching of the dye fluorescence. However, in the presence of specific target DNA, the adsorbed PNA could be desorbedfrom the GO surface by Waston-Crick specific hydrogen bonding with itscomplementary DNA, and then the initially quenched fluorescencerestored. Hence DNA was detected based on the fluorescence changebefore and after PNA-DNA hybridization in the solution of GO.In Chapter2, a novel fluorescent sensing platform for miRNAdetection was developed, which simply combined the fluorescencequenching efficiency of GO and the duplex-specific nuclease(DSN)-induced target recycling. Fluorophore-labeled DNA strands actingas probes were physically adsorbed onto the GO surface, leading tofluorescence quenching. In the presence of target miRNA, the DSNenzyme cleaved the probe DNA in DNA-RNA hybrid duplex into smallfragments and the miRNA was released from the duplex. Thus therecycling of the target miRNA was realized, producing numerous smallDNA fragments. After the introduction of GO into the sensing solution, astrong fluorescence emission was observed due to the weak interactionbetween the short DNA fragments and GO. With this approach, asub-picomolar detection limit of miRNA could be achieved within40min.What is more, this biosensor exhibited good sequence selectivity due tothe great sequence discrimination ability of DSN enzyme. The proposedsensor is sensitive, specific, simple and rapid, paving a way to themiRNA analysis.