Fluorescence Properties,Regulation Approach And Synthesis Mechanisms Of Self-assmbled Quantum Dots In Organisms

Quantum dots(QDs),owning to their unique optical and electronic properties,are promising for bio-imaging/bio-sensing/bio-diagnosis and optical device applications,but their large-scale production is still challenging.Compared to traditional chemical synthesis processes that typically involve high temperature,anaerobic,toxic reagents,surface modification reactions,biosynthesis has recently emerged as a green,sustainable approach to obtain QDs through self-assembly in living organisms.To obtain valuable QDs,precise regulation of QDs biosynthesis is of vital importance.In this dissertation,we investigated the fluorescence dynamics/regulation method/biosynthesis mechanism and application of the biosynthesized QDs in several model organisms by combining advanced molecular spectroscopic analysis,biomolecule techniques and theoretical calculation.The main contents and results of this dissertation are listed below:1.To characterize and assess the optical properties of QDs,we synthesized cadmium-selenium(CdSe)QDs in Candida utilis WSH02-08,and tracked and quantitatively characterized the developmental dynamics(photoactivation,photostable and photobleaching processes)of bio-QDs by combining fluorescence microscopy observation with image analysis.The photoactivation process was well fitted by stretched-exponential model(I = I0-Aexp-[t/?]?).The photobleaching data were well fitted by the bi-exponential form of"a1exp(-t/?1)+a2exp(-t/?2)".These findings shed light on the fluorescence properties of the bio-assembled QDs and provide a new way to screen bio-QDs and monitor the quality of QDs in vivo,which lay the foundation for their next-step applications.In addition,the phoactivation index/the maximum fluorescence intensity showed a good linear correlation to Cd concentration,implying a high potential to be used for Cd concentration detection.2.We prepared cadmium-selenium(CdSe)QDs in vivo using Candida utilis WSH02-08,and efficiently regulated the fluorescence properties of the bio-QDs through changing the precursor concentrations.Production of bio-QDs with high fluorescence intensity and long photostable lifetime were favoured at an elevated Cd content over Se,attributed to the formation of less Se(0)while more CdSe and CdS.In addition,the utility of the as-synthesized QDs for live-cell imaging without further modification was demonstrated.These findings will deepen our understanding on the mechanism of QDs fabrication in microorganisms,and provide an effective method for regulation of bio-QDs fabrication3.To accelerate bio-QDs production,which generally takes over 10 hours,we explored a new method to promote fast synthesis of bio-QDs by regulating the solution pH.Our results show that,while E.scherichia coli could barely synthesize QDs at neutral pH,lowing the pH to 4.5 endowed it an amazing synthetic ability,yielding uniform-sized CdSxSe1-x QDs with comparable fluorescence properties to their chemical counterparts.More intriguingly,an ultra-fast biosynthesis process(within 1 hour)was enabled.Lastly,we elucidated the biomolecule mechanisms,and demonstrated the utility of as-synthesized QDs for practical bio-imaging application in cancer cells and mice.Our work opens up a very simple yet effective approach for stimulation of bio-QDs synthesis,bringing bio-synthesized QDs a step closer to practical application.4.The bio-QDs synthesis is closely associated with the detoxification processes in living organisms,but the underlying mechanisms are unclear so far.In this work,the metabolic regulation on biotransformation and interaction of Se and Cd in Escherichia coli were invesigated.Raising the substrate(glucose)concentration significantly enhanced the retention of Cd and Se in E.coli and the dominant biotransformation products shifted from Cd3(PO4)2 to CdSxSe1-x QDs with excelluent fluorescence properties.This work sheds light on the QD synthesis-associated antagonism mechanism in E.coli,and provides valuable implications for bioremediation of metal contaminated environment and recycle of metal resouces from environment.5.The chemical structure,synthesis route and location organelle of bio-QDs were unraveled in this work by using E.coli as a model organism X-ray absorption spectroscopy and transmission electron microscopy were used to characterize the chemical structure of synthesized QDs,which showed apparent CdSe/CdS core-shell structure with and a phosphate-and organic selenium-rich shell.The QDs synthesis pathway was identified by gene modification assay and density functional theory calculations.Phosphate was found to act as Cd2+ taxi to assist selenol/thiol amino acid/protein in fabricating CdSe/CdS nanoparticles.Tracking the metallic substance sequestration and storage organelle by fluorescence microscope,Raman microscope and soft X-ray tomography revealed the presence of QDs at one end or both ends of each cell being aggregated in the form of metallicsome..6.We show that,by using Shewanella oneidensis MR-1 as dissimilar metal reducing bacteria model bio-producer,a high-rate,directed assembly of metallic nanoparticles was enabled through regulating the cellular transmembrane electron transfer.We obtained abundant CdSe nanoparticles in the cytoplasm after culturing the bacterium for four hours in a medium spiked with Na2SeO3 and CdCl2,much faster than previous biosynthesis processes.Intriguingly,a directed fabrication of CdSe over Se0 was enabled after obstructing the transmembrane electron transfer pathway by deleting the membrane-anchored CymA protein.Meanwhile,the dominant subcellular synthetic location shifted from periplasm/cell surface to cytoplasm,and the cellular detoxification ability raised significantly,indicating that cytoplasmic nanoparticle synthesis might function as an efficient detoxification mechanism.Our findings highlight the potential of exploiting the cytoplasmic detoxification and synthesis power in S.oneidensis MR-1 for high-rate,directed bio-fabrication of nanoparticles,and a further expending to other dissimilatory metal-reducing bacteria may hopefully lead to more diverse and fine-tunable nanomaterial bio-factories.