Functional Nucleic Acid Probe For Detecting ATP

Adenosine Triphosphate (ATP) is known as the energy metabolism of all life. Itis ubiquitous in all living cells and provide energy for the cell. It is playing animportant role in the central nervous system and peripheral nervous system signalsexpression. The content of ATP has important reference value in clinical testing forthe diagnosis of many diseases and also reflects the quantity of microorganisms inenvironment.A simple, rapid, sensitive and accurate detecting method for ATP is of particularimportance. It not only helps promoting the study of life processes, but also haspractical applications in clinical diagnosis, drug analysis, drug development and otherfields. Nucleic acid probe has the advantage of real-time, fast, stable, intuitiveanalysis and ultra-sensitive detection. Analysis and detection of ATP combiningnucleic acid probe has extremely broad space for development and applicationprospects in many fields such as medicine, environment, food and life sciences. Thethesis, based on the colorimetric, fluorescence, confocal fluorescence imaging andsurface-enhanced Raman spectroscopy methods, we have designed a series offunctional nucleic acid probes for detecting ATP.(I) A label-free colorimetric method to detect ATP catalyzed by deoxyribozyme(DNAzyme) was designed. With the addition of ATP, the release of single DNA andthe G-quadruplex structure was formed, then the concentration of ATP was detectedby UV-Vis spectroscopy.With the effect of the catalytic activity of DNAzyme, a ultrasensitive andselective detecting methods for ATP based on G-quadruplex/hemin-ATP DNAzyme(GHAD) was proposed. The hairpin structure that included the HRP-mimickingDNAzyme in caged was designed. ATP can trigger the hairpin conformation changeand form GHAD complexes which can catalyze the H2O2oxidation form ABTS2-toABTS-. The strategy has low background-signal which can achieve sensitive andselective detection of small molecular by colorimetric method. The linearconcentration range was0.5-2000μM for ATP with a detection limit of0.1μM.Meanwhile, it can be applied to detect the ATP in serum samples.(II) In-situ growth of silver nanoparticles of the free single DNA strand inSWCNTs, a sharply change on the signal of SERS can be obtained. Based on it, anovel homogeneous aptamer recognizing and detecting method for ATP by SERS was established. The linear concentration range was5-100nM for ATP with a detectionlimit of5nM. The nanoprobe can detect ATP in the serum samples with a detectionlimit of10nM. The proposed method show good selectivity for ATP.(III) A highly sensitive and selective fluorescence aptamer biosensors for thedetermination of ATP was developed. Binding of a target with splitting aptamerslabeled with pyrene molecules form stable pyrene dimer in the γ-cyclodextrin (γ-CD)cavity, yielding a strong pyrene excimer emission. We have found that inclusion ofpyrene dimer in γ-CD cavity not only exhibits additive increases in quantum yield butalso fluorescence lifetime of the excimer. With an anti-ATP aptamer, the approachexhibits excimer fluorescence response toward ATP with a maximumsignal-to-background ratio of32.1and remarkably low detection limit of80nM inbuffer solution. Moreover, due to the additive fluorescence lifetime of excimerinduced by γ-CD, time-resolved measurements could be conveniently used to detectas low as0.5μM ATP in blood serum quantitatively.(IV) I-motif/nanoflares for detecting ATP in lysosome was designed. In pH=7.4buffer solution, fluorescence enhancement couldn’t be observed. In pH=5.2buffersolution, with the addition of ATP, the fluorescence emission increased. Both H+andATP were served as triggers to disassemble the complex, the flare strand wasdisplaced and liberated from the AuNPs, fluorescence recovered. By combiningstructure-switchable i-motif sequence and high recognition ability of ATP aptamer,subcellular sensing and imaging of ATP in lysosome can be achieved. The overlap ofred fluorescent probes and green fluorescent of commercial lysosomal dyes showncolocalization with high efficiency.