Determination Of Adenosine Triphosphate Based On Chimeric Aptamer And Non-labeled Fluorescence Analysis Method

Adenosine triphosphate(ATP)is a high-energy phosphate compound and the main energy currency of life.It participates in regulating various biological processes and biochemical pathways,which plays a critical role in the maintenance of the normal function of the organism.Therefore,it is of great significance to establish accurate and sensitive methods for ATP in biochemical research and clinical diagnosis.Among many strategies for ATP,fluorescence-based methods have become one of the most important strategies due to its advantages of high sensitivity,good selectivity,multiple characteristic parameters and wide dynamic range.Moreover,the reports of aptamers make nucleic acids a promising tool for analytical applications.So far,aptamer-based sensors for ATP have been used in lots of researches such as the delivery of ATP-induced anticancer drugs.Combined the interactions between fluorescent dye and nucleic acid ligands,bifunctional DNA molecules were designed and optimized,and three novel label-free fluorescence analytical methods for ATP were developed by further.Chapter 1 IntroductionIn sequence,the significance and methods of the ATP detection,the properties and applications of the nucleic acid aptamers,the interactions between fluorescent dyes used in this study and nucleic acid ligands and aptamer-based fluorescent analytical methods for ATP were introduced.Finally,the research contents and objective were presented.Chapter 2 Design and optimization of bifunctional chimeric aptamers with berberine as the probeCurrently,hundreds of aptamers have been reported and aptamer-based biosensors become much attractive in analytical applications.In the traditional study of ATP detection,single-function aptamers were often used.To improve the sensitivity for ATP,designing a novel multipurpose aptamer probe can be a promising strategy.In present work,combined the characters of berberine binding-aptamer(BBA)and ATP binding-aptamer(ABA),the bifunctional chimeric aptamers were designed and optimized.This chimeric aptamers,BBA-ABA,not only had the ability of recognizing ATP but also could capture berberine and make the fluorescence intensity enhanced.It was observed that the chimeric aptamer was hydrolyzed by Exo I leading to the release of berberine in the absence of ATP,and making the fluorescence intensity of this system weakened.While when ATP was present,the ABA aptamer on 3' prime of BBA-ABA bound specifically to its target,switching its secondary structure from random coil to G-quadruplex,which can resist attack from Exo I.This made the fluorescence probe berberine remain bound to BBA,keeping its enhanced fluorescence intensity.Inspired by these discoveries,the designed chimeric aptamer was used to develop an Exo I-assisted fluorescence sensor for the determination of ATP.The sensitivity of the designed chimeric aptamer was 5.5 times higher than that of the single ATP aptamer.The detection limit(3?)of this method for ATP was estimated to be 123 nM,and this method was highly selective for ATP.Finally,the feasibility of the proposed method was evaluated in real biological samples,and the ATP spiked in human serum was detected with satisfactory results.Chapter 3 Chimeric aptamers-based and MoS2 nanosheet-assisted fluorescence polarization biosensor for ATP detectionCompared with fluorescence spectroscopy-based methods,fluorescence polarization(FP)strategy is less sensitive to confounding elements such as photobleaching and instrumental parameters,possessing the ability of anti-enviroment disturbance.However,FP usually has the challenge of developing a general method for mass amplification in order to deal with the problem that most of molecular weights are too small to make detectable FP changes.This study developed a chimeric aptamer-based and MoS2 nanosheet-enhanced fluorescence polarization biosensor for ATP.It was observed that in the absence of MoS2 nanosheets the hydrolysis of Exo I was hindered becauae of the addition of ATP.The changes of FP signal caused by the addition of ATP was not obvious,which would limit the application of FP method in ATP detection.The subsequent addition of MoS2 nanosheets highly magnified the FP changes before and after the addition of ATP.The detection limit(3?)of this FP strategy for ATP was as low as 34.4 nM,which was 3.5 times lower than that of chapter 2.This FP strategy was highly sensitive and selective for ATP.Finally,the feasibility of the proposed FP method was evaluated in real biological samples,and the ATP spiked in human serum was detected with satisfactory results.Chapter 4 Based on chimeric aptamer with berberine as the reference and label-free ratiometric fluorescence method for ATP detectionCompared with single-wavelength measurements,ratiometric fluorescence methods have the advantage of avoiding the effect brought by the fluctuation of excitation light intensity.This study optimized a chimeric aptamer with ThT as a probe and berberine as a reference based on the aptamer of chapter 2.This chimeric aptamer also not only could capture berberine but also recognize ATP.It was observed that berberine and ThT was respectively captured by BBA and ABA after the addition of berberine and ThT,making their fluorescence intensity enhanced.In the presence of ATP,the ABA aptamer on 3'prime of chimeric aptamer bound specifically to its target,causing the release of ThT and making the fluorescence intensity of ThT highly weakened.However,the addition of ATP had no significant effect on the interaction between berberine and chimeric aptamer,keeping the enhanced fluorescence intensity of berberine.Inspired by these discoveries,the optimized chimeric aptamer was used to develope a label-free ratiometric fluorescence strategy for ATP,with berberine as the reference and ThT as the probe.The detection limit(3?)of this ratiometric fluorescence strategy for ATP was as low as 24.8 nM,which is lower than that of chapter 2 and 3.Compared with the single-wavelength detection method of single aptamer reported in the literature,the ratio method of chimeric aptamer not only eliminated the effect brought by the fluctuation of excitation light intensity,but also improved the detection sensitivity.Finally,the feasibility of the proposed ratiometric fluorescence method was evaluated in real biological samples,and the ATP spiked in human serum was detected with satisfactory results.Chapter 5 ConclusionSummarize the whole paper and make suggests for future research.