The Design Of Nucleic Acid Aptamer/Berberine Fluorescence System And Its Analytical Application

Chapter 1:Firstly, the definition, selection and properties of nucleic acid aptamer are introduced briefly. Recent progress in aptamer-based sensing technology is reviewed in detail. Finally, the research background and main contents of the thesis are outlined simply.Chapter 2:A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new fluorescent probe/aptamer system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.Chapter 3:A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N= 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust and stable in rough conditions. This strategy can be generalized for detection of a variety of targets through selecting an appropriate aptamer.Chapter 4:A simple, rapid and label-free fluorescent aptasensing platform was fabricated for the detection of Hg2+ion in aqueous solution using Hg2+-aptamer as the recognition element and berberine as the fluorescence probe. Various factors including the concentration of berberine and aptamer, pH effect and reaction time were investigated in detail. Under the optimal experimental conditions, The fluorescence intensity of the sensing system displayed a calibration response for Hg2+ ion in the range of 2 μM to 10 μM with a detection limit of 8.0 nM (S/N=3). This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of Hg2+ion in blood serum samples with the recovery range of 96.3%-100.5%.