Detections Of NAD(H) And Its Conformation Based On Biomimetic Molecular Receptor Nanopore

Biological systems contain a variety of complex structures and functions,such as intrinsic heterogeneity and catalytic conversion.To further clarify the various physiological mechanisms in such complex systems,innovation in single-molecule biotechnology is imperative.Contrary to traditional bulk techniques,single-molecule nanochannel technology can track individual biomolecules in space and time.More importantly,this method has the unique function to acquire fingerprint information of multiple molecular at the same time.Therefore,it is suitable for high-throughput multiplex analysis of complex bio-fluids and components with minor differences.Herein,the biomimetic molecular receptor,octakis-(6-amino-6-deoxy)-?-cyclomaltooctaose,has embedded in the mutated?-hemolysin nanopore,which has been used as a recognition sensor to detect nicotinamide adenine dinucleotides(NAD(H))and its conformations at the single-molecule level.Details of the studies in this thesis could be summarized as follows:1.Biomimetic molecular receptor nanopore for simultaneous quantifications of NAD⁺and NADHInspired by biological systems and influenced by the simulation results of the molecular size of NAD(H),we designed and synthesized a biomimetic NAD⁺/NADH molecular receptor,octakis-(6-amino-6-deoxy)-?-cyclomaltooctaose(am₈?CD),and am₈?CD was non-covalently bound to the mutant?-HL(M113R)₇ nanopore to construct a novel single-molecule biosensor.The single-molecule measurement possesses high selectivity and signal-to-noise ratio,allowing to simultaneously recognize and detect NAD⁺and NADH without any modification.On this basis,we further monitored the NAD⁺conversion process catalyzed by Saccharomyces cerevisiae alcohol dehydrogenase,which provides a new strategy for the detection of enzyme activity and the screening of enzyme inhibitors.2.Study the conformational changes of NAD(H)with biomimetic molecular receptor nanoporeBased on molecular simulation,molecular docking,and the inherent conformational preference of NAD⁺and NADH,the different platforms of characteristic signals for NAD(H)generated by the?-HL(M113R)₇-am₈?CD nanopore were analyzed corresponding to the two conformations of NAD(H),namely the folded form and the unfolded form.On this basis,organic solvent and voltage dependence experiments were carried out to explore the influence of methanol and applied voltage on the conformational changes of NAD(H).The experimental results show that the introduction of methanol molecules can destroy the stacking of the two aromatic heterocyclic rings of NAD(H),thereby promoting the transformation of NAD⁺and NADH from the folded form to the unfolded form.The high voltage can drive NAD(H)to occur unfolding process and pass through the nanopore in unfolded conformations.