ABSTRACT
As a typical representative of endocrine-disrupting chemicals (EDCs), bisphenol A (BPA) is a common persistent organic pollutant in the environment that can induce various diseases even at low concentrations. Herein, the magnetic Fe3O4-Au@Ag@(Au@Ag) nanocomposites (CSSN NCs) have been prepared by self-assembly method and applied for ultra-sensitive surface-enhanced resonance Raman scattering (SERRS) detection of BPA. A simple and rapid coupling reaction of Pauly's reagents and BPA not only solved the problem of poor affinity between BPA and noble metals, but also provided the SERRS activity of BPA azo products. The distribution of hot spots and the influence of incremental introduction of noble metals on the performance of SERRS were analyzed by a finite-difference time-domain (FDTD) algorithm. The abundance of hot spots generated by core-shell-satellite structure and outstanding SERRS performance of Au@Ag nanocrystals were responsible for excellent SERRS sensitivity of CSSN NCs in the results. The limit of detection (LOD) of CSSN NCs for BPA azo products was as low as 10-10 M. In addition, the saturation magnetization (Ms) value of CSSN NCs was 53.6 emu·g-1, which could be rapidly enriched and collected under the condition of external magnetic field. These magnetic core-shell-satellite NCs provide inspiration idea for the tailored design of ultra-sensitive SERRS substrates, and thus exhibit limitless application prospects in terms of pollutant detection, environmental monitoring, and food safety.
ABSTRACT
Current strategies to improve the throughput of continuous directed evolution technologies often involve complex mechanical fluid-controlling system or robotic platforms, which limits their popularization and application in general laboratories. Inspired by our previous study on bacterial range expansion, in this study, we report a system termed SPACE for rapid and extensively parallelizable evolution of biomolecules by introducing spatial dimensions into the landmark phage-assisted continuous evolution system. Specifically, M13 phages and chemotactic Escherichia coli cells were closely inoculated onto a semisolid agar. The phages came into contact with the expanding front of the bacterial range, and then comigrated with the bacteria. This system leverages competition over space, wherein evolutionary progress is closely associated with the production of spatial patterns, allowing the emergence of improved or new protein functions. In a prototypical problem, SPACE remarkably simplified the process and evolved the promoter recognition of T7 RNA polymerase (RNAP) to a library of 96 random sequences in parallel. These results establish SPACE as a simple, easy to implement, and massively parallelizable platform for continuous directed evolution in general laboratories.
Subject(s)
Bacteriophages , Agar/metabolism , Bacteria/genetics , Bacteriophages/metabolism , Escherichia coli/genetics , Promoter Regions, GeneticABSTRACT
To investigate the expression and localization of various functional domains of ORF1 polyprotein and ORF3 protein of hepatitis E virus in host cells, the coding sequences of the various functional domains (RdRp, HEL, MET, PLP, X) of ORF1 were separately cloned into pcDNA3. 1-GFP vectors for constructing the recombinant plasmids which were verified by enzyme digestion and sequencing. The exact expression of the fusion proteins were detected by Western Blot, and the distribution and localization were observed by the laser scanning confocal microscope(LSCM). In huh7 cells, GFP-RdRp proteins were found mainly in the nuclei, GFP-HEL proteins were distributed vesicularly around the nucleus, GFP-MET proteins were distributed granularly both in the nuclei and the cytoplasm, GFP-PLP proteins had polar distribution around the nucleus, and unknown GFP-X proteins were distributed uniformly both in the nuclei and the cytoplasm. Different localization of these proteins verified the previous data obtained from in vitro studies, providing a support for further research on the biological functions of various proteins coded by HEV genome.
