| Background Self-assembled nanostructures can improve detection sensitivity in enzyme-linked immunosorbent assay by mediating the increase of enzyme molecules number and the efficient immobilization of antibodies. Molecular self-assembly is a process that molecules spontaneously form a highly ordered structure. It widely exists in nature and is the main organization form of life. Self-assembly approach is attractive for novel functional nanodevice fabrication, due to their uniformity, versatility and simplicity of preparation. However, self-assembly is a spontaneous process, mainly governed by thermodynamics, therefore, the process is hard to control. Furthermroe, the components with different self-assembling domain cannot assemble together, resulted the self-assembled nanostructure lack of funtions. Both limit the increase of detection sensitivity. Thus, the development of a straightforward method to construct self-assembled superstructure of controllable size and multiple functions is highly desirable.Purpose We want to establish novel controllable self-assembly strategies to construct homogeneous nanostructure with sophisticated functions. And further exploit to build complex biological nano-devices for a broad range of biomedical applications.Methods Here we showed two novel controllable self-assembly strategies for construction of multi-functional nanoparticle assemblies and bifunctional ferritin nanoparticls in vitro and in vivo respectivily. First, we present a strategy of size-controlled self-assembly in which the number of interacting building blocks could be limiting by rationally controlling the number of self-assembling interaction sites on the surface of each building block. In addition, we chose a specific biomolecular interaction, biotin-streptavidin interaction, as the driving force to mediate the interaction during assembling process. Based on this principle, a size-controlled enzyme nanocomposite(ENC) was constructed by self-assemble of streptavidin-labeled horseradish peroxidase(SA-HRP) and autobiotinylated ferritin nanoparticles(bFNP). The ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in sensitivity of immunoassay. Secondly, we explored the controllable asymmetric self-assembly strategy in vivo by using the method of synthetic biology and present an one-step preparation of multifunctional and asymmetric protein particles by adjusting the metabolic network of Escherichia coli.Results In the study of ENCs with tunable size, SA-HRPs were used to titrate bFNPs in a series of ratios and the size-controlled self-assembly can be achieved by regulating the stoichiometric ratio of the two building blocks during assembling process. The results of characterizations indicated that significant changes of sizes were found depending on the assembly ratio. In the presence of saturated or excess SA-HRP, the coating of SA-HRP prevented the aggregation of bFNPs, resulting in the construction of ENCs that contained only one bFNP in each structure. In the presence of insufficient SA-HRP, bFNPs shared SA-HRP with each other, resulting in the aggregation of bFNPs and a concomitant increase of the size of ENC. For the research of self-assembly in vivo, the co-expression vector was construct as expected and the different functional subunits were assembled into one protein cage simultaneously, forming multi-functional ferritin nanoparticles.Conclusions (1) We developed a size-controlled self-assembly strategy which achieved by limiting the number of interacting nanoparticles through limiting the number of interaction sites on the surface of these nanoparticles and achieved size-controlled enzyme nanocomposite for highly sensitive detection. (2) The multifunctional nanoparticles were achieved with one-step process by the differential expression of ferritin subunits with different functional ligands and the mixed assembly of different functional protein subunits in vivo. |
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