Study On The Interaction Between Multifunctional Zero-Dimensional Quantum Dots And Proteins And Their Mechanism

On account of its excellent biocompatibility and excellent physical and chemical properties,zero-dimensional quantum dots have been widely applied in many biomedical fields,such as in vivo/in vitro imaging,disease diagnosis,targeted drug delivery and clinical treatment.However,the current functions and effects of zero-dimensional quantum dots are still difficult to support their entry into deeper and safer clinical applications.This is mainly due to the fact that since the advent of quantum dots,researchers have mostly devoted themselves to studying the biological effects and toxicological mechanisms of zero-dimensional quantum dots in a relatively single and limited way in vivo/in vitro.The subtle and non-negligible physicochemical interaction between zero-dimensional quantum dots is often difficult to pay attention to.Nanomaterials such as quantum dots are prone to complex interactions with biological protein molecules,whether in complex organisms or in a single,controlled environment in vitro.This process is often difficult to avoid the impact of the original biological effects of nanomaterials and the migration,transformation and deposition processes in vivo and in vitro,and may lead to larger toxicological reactions in cells,tissues,organs and even individual organisms.At present,the biological understanding of different types of zero-dimensional quantum dots is not systematic and in-depth,especially the research on the physicochemical binding effect and binding mechanism between quantum dots and biological protein macromolecules（such as bovine serum albumin,human serum albumin,trypsin,etc.）is still insufficient.Therefore,it is urgent and of great significance to explore the interaction mechanism between biological protein molecules such as bovine serum albumin,human serum albumin,trypsin and zero-dimensional quantum dots based on thermodynamic and kinetic theory through a variety of spectroscopy and molecular simulation strategies.This paper from the protein molecular level research discussed the black phosphorus quantum dots,molybdenum disulfide quantum dots and titanium carbide quantum dots of the three different structure and physical and chemical properties of zero-dimensional nanoparticles respectively with bovine serum albumin and human serum albumin and trypsin these three different function,the structure of the interaction mechanism between protein biological macromolecules.This paper is divided into six chapters:Chapter 1:An overview of the research status of the structure,properties,biological effects and toxicology of black phosphorus quantum dots（BPQDs）,molybdenum disulfide quantum dots（Mo S₂ QDs）,and titanium carbide quantum dots（Ti₃C₂ QDs）.And an overview of the structure and function of the three protein biomacromolecules（Bovine serum albumin（BSA）,Human serum albumin（HSA）,Trypsin,respectively）and the interaction of various types of nanoparticles with these three protein molecules.And the current research strategies of various types of nanoparticles and protein molecules are briefly described.Chapter 2:The affinity interaction between BPQDs and BSA is systematically and deeply characterized,in order to interpret the conformational changes of BSA affected by BPQDs in a detailed and systematic manner.Through a variety of fluorescence strategies and molecular docking results,it was revealed that the intrinsic fluorescence of BSA was spontaneously quenched by BPQDs in a static quenching mechanism based on van der Waals interaction-dominated non-chemical bond induction.Moreover,BSA and BPQDs were strongly associated to form ground-state complexes at a molar ratio of 1:1,so that BPQDs could be tightly bound to the Site I domain of BSA with low binding.Chapter 3:The binding mechanism of transition metal disulfide Mo S₂ QDs and HSA was systematically and deeply explored.The effect of Mo S₂ QDs on HSA secondary structure and fibrillary formation in physiological buffer solution was comprehensively analyzed by multi-spectral strategy（including three-dimensional fluorescence,synchronous fluorescence,circular dichroism,infrared spectroscopy）and laser confocal fluorescence microscopy.The fluorescence temperature-changing strategy,molecular simulations and electrochemical techniques synergistically demonstrate that Mo S₂ QDs spontaneously generate strong non-bonding interactions with HSA under the combined promotion of hydrogen bonding and van der Waals forces.In addition,Mo S₂ QDs firmly binds to the Site I domain of HSA with a stoichiometric ratio of 1:1 and a low binding energy under the mechanism of spontaneous static fluorescence quenching.Under this strong,relatively stable and specific binding site force,Mo S₂ QDs induced a significant increase in the random curl structure in HSA in a concentration-dependent manner and gradually destroyed theα-helix secondary structure of HSA,making the internal group structure of HSA become more hydrophilic and looser.This process also revealed that Mo S₂ QDs accelerated the thermal denaturation process of HSA by destroying and reducing the biothermal stability of HSA.Furthermore,the presence of S atoms in Mo S₂ QDs interferes or even inhibits the formation of disulfide bonds during HSA protein fibrosis,thereby significantly inhibiting HSA aggregation.Fluorescence microscopy imaging demonstrated the irreversible effects of Mo S₂QDs on the formation of HSA fibrosis,implying the potential application of Mo S₂ QDs in the prevention of HSA amyloidosis.These results dissect the biological effects of Mo S₂ QDs at the molecular biology level,laying a foundation for the safer and more friendly biological applications of Mo S₂ QDs in nanomedicine-related fields.Chapter 4:The binding effect of inorganic non-metallic BPQDs and trypsin was comprehensively and deeply studied.Through a variety of fluorescence strategies and molecular docking results,it was revealed that the intrinsic fluorescence of Trypsin was spontaneously quenched by BPQDs in a static quenching mechanism under the non-chemical bond-induced van der Waals interaction.And BPQDs bind firmly to the inactive residue domain of Trypsin with a1:1 stoichiometric ratio and low binding energy.Exposure of trypsin to a physiological buffer solution rich in BPQDs significantly induced and disrupted the secondary and tertiary protein structures of Trypsin.The calculation of Michaelis constant and related kinetic parameters of the BPQDs-Trypsin system and the results of molecular simulation experiments revealed that BPQDs significantly affected the specific cleavage performance of proteases based on the non-competitive inhibition of Trypsin activity.Gel electrophoresis experiments and microscopic imaging experiments of Hela and A549 cell digestion visualized the destructive killing effect of BPQDs on Trypsin cleavage activity,thereby protecting HSA,Hela cells and A549 cells from trypsin digestion.The results of this chapter reveal the deep mechanism of the interaction between BPQDs and protease,the exact binding position and the effect on the activity of Trypsin,paving the way for the safe nanobiotic application of BPQDs in the treatment of diseases and other aspects.Chapter 5:A method to construct transition metal carbide Ti₃C₂ QDs by hydrothermal method is proposed.The synthesized Ti₃C₂ QDs exhibited a smooth and plump fluorescence emission peak at 475 nm under the excitation wavelength of 370 nm.Furthermore,the Ti₃C₂QDs have good biocompatibility and can be applied to blue fluorescence imaging of cells based on their successful internalization by Hela cells by endocytosis.In this chapter,the interaction mechanism between the newly constructed Ti₃C₂ QDs and trypsin was also discussed through various spectroscopic strategies.The results demonstrate that the intrinsic fluorescence of Trypsin is spontaneously quenched by the static quenching mechanism of Ti₃C₂ QDs under the non-chemical bond-induced van der Waals interaction force-dominated.And Ti₃C₂ QDs firmly bound to the active residue domain of Trypsin with a 1:1 stoichiometric ratio and low binding energy.Exposure of trypsin to a solution enriched with Ti₃C₂ QDs significantly reduced the thermal stability of Trypsin in aqueous solution.At the same time,gel electrophoresis experiments and Hela cell digestion microscopic imaging experiments both verified the destructive killing effect of Ti₃C₂QDs on the enzyme cleavage activity of Trypsin.These conclusions explored the interaction mode of transition metal carbide Ti₃C₂ QDs with Trypsin,the lowest binding energy domain and the effect on Trypsin cleavage activity.The close connection of chemistry,materials science and nanobiomedicine provides crucial information for the future research of Ti₃C₂ QDs into deeper and safer biological applications.Chapter 6:Summary and outlook.