Studies Of Novel, Label-free Fluorescent Biosensing Technology For The Detection Of Small Biomolecules

As a high energy compound, adenosine triphosphate(ATP), exsists in almostall living organisms. It participates in the metabolism processes of living cells,such as cellular functions, membrane transport, compound synthesis and decomposition, and so on. The content of ATP in organisms is of great importance tothe metabolism and death of cell. Moreover, the occurrence and development ofmany diseases, such as cardiovascular, Parkinson and Alzheimer, have been verified to be connected with the anomalous change of the level of ATP. Nicotinamide adenine dinucleotide(NAD+) can serve as the cofactor of many enzymes, aswell as charge carrier, which transfers protons in metabolic reactions. It plays acritical role in cell proliferation, death, regulation of transcription, DNA repair,calcium homeostasis and caloric-restriction-mediated lifespan extention.Label-free fluorescent sensing strategy is convenient, fast and highly sensitive,which has attracted more and more interest of the researchers. Thus a series oflabel-free fluorescent sensing strategies has been developed, used for the analysis ofsmall biological molecules including ATP and NAD+, in this thesis. The details aredescribed in following chapters:In Chapter2, we developed a novel label-free fluorescent strategy based onnicking endonuclease signal amplification(NESA) and N-Methyl mesoporphyrin(NMM)-responsive G-quadruplex formation for the detection of ATP. The nick in thespecially designed DNA duplex can be sealed by T4DNA ligase in the presence of ATP.The following replication and strand displacement yield the duplex that contains thefull recognition site for nicking endonuclease，followed by NESA process. The nickedstrand can fold into G-quadruplex structure in the presence of K+and selectively bindswith NMM, yielding a significant fluorescence signal. Under the optimal conditions,the fluorescent assay strategy showed a dynamic response to ATP in the concentrationrange from10nM to1000nM and a detection limit of0.8nM, which comparedfavourably with most of the previous ATP assay methods. In addition, the proposedstrategy exhibited superior selectivity towards ATP over other nucleosides and itsanalogues as well as simplicity and excellent sensitivity.In Chapter3, a simple lable-free fluorescent sensing scheme for sensitive andselective detection of NAD+has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation.This method can realize indirect detection ofNAD+, beginning with the ligation reaction trigged by E.coli DNA ligase with theassistance of NAD+. This label-free approach can detect0.5nM of NAD+with highselectivity against other NAD+analogs, while remains superior properties offluorescent sensors. It’s cost-effective, sensitive and fast.In Chapter4, based on graphene oxide(GO), a simple lable-free fluorescentaptasensing scheme has been developed. GO can strongly absorb single-stranded DNA,but only weakly absorb SYBR Green I(SG) stained dsDNA. Thus in the later case, GOcannot effectively quench the fluorescence emission of the fluorescent dye. In thepresence of ATP, the divided aptamers bind with the target and make the formed dsDNA complex intercalated with SG, yielding significant fluorescence enhancement.The proposed approach is fast, simple and convenient with the detection limit of0.5μM.