Construction Of Biosensing System For MRNA Detection Based On Nucleic Acid Probe And Nanomaterial Of Graphene

Biosensor holds great potential in medical analysis and scientific research due to its advantages of high specificity,high detection speed,low cost,high sensitivity and the ability to be used in complex system.Nucleic acid probes and nanomaterials have been widely used in the field of biosensors and exhibit great achievements.Therefore,they can provide rich sensors design inspirations.This master's thesis is based on advantages of nucleic acid probes and graphene nanomaterials in biosensing and biochemical analysis applications,combining with the enzymatic signal amplification methods and multi-site real-time detection methods for sensitive and quantitative analysis of mRNA and real-time imaging of live cells.The developed methods are high sensitivity,specificity and low cost.The detailed contents are described as follows.p21,a cell cycle regulatory protein,plays a very important role in the regulation of cell proliferation and tumorigenesis.As an antitumor gene,the level of p21 mRNA is significantly decreased in the tumor tissues and cell lines compared with normal cells.Thus,p21 mRNA can be used as a biomarker for tumor diagnosis,clinical therapy and prognosis evaluation.In chapter 2,we developed a new graphene oxide nansheet(GONS)based fluorescence method for mRNA assay with duplex-specific nuclease(DSN)-assisted signal amplification.Following hybridization between target mRNA and its complementary DNA probe,the ssDNA in the DNA/mRNA hybrids is selectively cleaved by DSN to produce small fragments.The released mRNA then initiates another cycle of hybridization and DSN digestion.In this manner,each mRNA molecule can specifically trigger many cycles of hybridization and DNA cleavages to produce numerous small NDA fragments.The short DNA fragments can exhibit strong fluorescence signal due to the weak adsorption of GONS to them.This fluorescence assay for mRNA with increased selectivity has a 1 fM detection limit under optimal conditions.Tumor-related mRNA has been regarded as a biomarker for tumor burden and malignant progression.Detection of tumor-related mRNA can provide powerful tools for identifying cancer cells at the genetic level in early stage.Therefore,it is important to develop biosensors to quantitatively and multiply detect mRNA expression levels.Here,we present a detailed protocol for in situ multiple fluorescence monitoring of p53 and p21 mRNA in HepG-2 liver cancer cells by using reduced graphene oxide nanosheet(rGONS)and DNA probes.FAM-labeled p21 probe and Cy5-labeled p53 probe are used to construct p21&p53@rGONS sensing platform through ?-? stacking between probe and rGONS,resulting in ‘fluorescence off' that is conjugated to the probe,and subsequent recovery of the fluorescence upon addition of target mRNA.On the basis of the fluorescence ‘off/on' switching,simultaneous sensing and imaging of p21&p53 in vitro and in situ have been realized through fluorescence and confocal microscopy techniques.In this work,we describe the detection of p21&p53 in vitro,the cytotoxicity of rGONS,the uptake mechanism of rGONS,and realize intracellular p21&p53 real-time in situ imaging in HepG-2 cells during cisplatin-induced.