Development Of Novel Fluorescent Biosensing Technology For The Detection Of DNA And Adenosine5’-triphosphate

Nucleic acids are routinely used as biomarkers to help diagnose pathogenicinfections and genetic disorders. The development of different methods for rapid,economic, sensitive and selective DNA detection is of great importance for theclinical diagnosis, gene expression analysis and biomedical studies.Adenosine5’-triphosphate (ATP) is a multifunctional nucleoside triphosphate often used as auniversal energy storage molecule in all living organisms. It plays a critical role inthe regulation of cellular metabolism and biochemical pathways in cell physiology.ATP has also been widely used as an indicator of living organisms for cell viabilityand cell injury. Besides, ATP depletion is related to pathogenesis such as ischemia,Parkinson’s disease, and hypoglycemia. Therefore, the developement of novelbiosensing methods for sensitive and selective assay of DNA and ATP is highlydesirable in biochemical study and clinical diagnosis.Nowadays, Due to its unique DNA absorbing ability and universal quenchingproperties for fluorophores, graphene oxide (GO) has been widely applied inbiomolecular detection. GO-based fluorescent biosensors show several advantagescompared with the traditional ones, including improved assay sensitivity, low costand enhanced signal to background ratio and so on. These advantages reveal thatGO-based fluorescent biosensors hold great potential in biomolecular detection.A label free detection strategy can provide a fast and cost-effective assay whichhas gained much interest. N-methyl porphyrin propionic acid IX (NMM) and SYBRGreen I (SG) are the most frequently used DNA interacting dye in label free assays.They have been widely applied for molecular detection because of their favorablephotophysical properties, thermal stability, and high sensitivity.According to the above considerations and the reported literatures, thisdissertation focuses on developing several novel label-free biosensors for DNA andsmall biological molecule detection. The detailed methods are described as follows:In Chapter2, a label-free sensitive fluorescent DNA biosensor was presentedbased on isothermal circular strand-displacement polymerization reaction (ICSDPR)combined with GO binding. This biosensor relies on the hybridization of target DNAwith hairpin probe as the prerequisite for ICSDPR and the preferential binding ofGO to stained single stranded DNA (ssDNA) over double stranded one to improve the signal to background ratio. By combining the properties of GO and SG with theICSDPR amplification, this fluorescent DNA biosensor displays a wide dynamicrange and a low detection limit of4pM. The proposed strategy is simple,cost-effective and sensitive, which might provide a promising method of choice forconvenient DNA detection.In Chapter3, taking G-quadruplex formation as signal readout, we haveconstructed a label-free fuorescent biosensor for the assay of ATP based on theATP-dependent enzymatic reaction (ATP-DER) and exonuclease III (Exo III)-aidedselective digestion.This strategy relies on the principle that Exo III shows differentcleavage capacity for a DNA substrate in the absence and presence of ATP.thenicked DNA duplex would be digested by Exo III, resulting in the formation ofG-quadruplex selfassembly with NMM and K+. The results revealed that the methodhas a low detection limit of2nM. The proposed strategy was simple, label-free andshowed high selectivity to ATP that could distinguish ATP from its analogues.In Chapter4, a simple, amplification-free and sensitive fluorescent biosensorfor ATP detection was developed based on the ATP-dependent enzymatic reaction(ATP-DER) and GO binding. In this assay, The formed nick could be sealed by T4DNA ligase in the presence of ATP, and the resulting unnicked DNA duplex wasresistant to thermal denaturation. Whereas the ligation reaction would not happen inthe absence of ATP, and the nicked DNA duplex was subject to thermal denaturation,which leads to the separation of two half DNA probes and template. After additionof GO, the fluorescence of the nicked DNA duplex was greatly quenched, whereasefficient fluorescence quenching did not occur to the unnicked DNA duplex and highfluorescence intensity was obtained. The results revealed that the method allowedsensitive quantitative assay of ATP with a wide linear response and a low detectionlimit of0.3nM. The proposed strategy was simple, amplification-free and showedhigh selectivity to ATP that could distinguish ATP from its analogues.