Ratiometric sensing of metabolites using dual-emitting ZnS:Mn2+ quantum dots as sole luminophore via surface chemistry design.

We herein present an effective and versatile platform for ratiometric sensing of metabolites using intrinsically dual-emitting ZnS:Mn2+ quantum dots (QDs) as sole reporter. To avoid notoriously non-specific interactions, a special triple-layer "filter screen" around the inorganic QD core is rationally constructed, which is made of oleic acid, cetyltrimethyl ammonium bromide and bio-enzymes. In the presence of the analytes, the in-situ enzymatic H2O2 molecules diffuse and pass through the "filter screen" along the molecule interspace, which then reacts with the inorganic core and leads to more dramatically quenching of the Mn2+ emission. The ratiometric signal readout is so distinct that can be observed by naked eyes (from orange to violet). In contrast, various coexisting bio-molecules, due to larger size, are well prevented from penetrating the filter screen by steric hindrance effect. So, various potential interfering substances do not disturb the assay. Under optimal conditions, five kinds of the corresponding substrates, namely glucose, cholesterol, lactate, xanthine and uric acid are well quantified by the emission intensity ratio of I470/I615, and the linear ranges are 0.1-200µM, 0.1-200µM, 1-200µM, 1-200µM and 1-200µM, respectively. The detection limits can even reach quasi-picomole levels. Because of favorable analytical performances (excellent selectivity, appropriate sensitivity and broad linear range), the proposed system can direct assay the analytes in blood without any sample pre-treatment.

Recombinant expressions of sweet plant protein mabinlin II in Escherichia coli and food-grade Lactococcus lactis.

Sweet plant proteins, which are safe, natural, low-calorie sweeteners, may be suitable replacements for sugars in the food and beverage industries. Mabinlin II, a sweet plant protein, shows the most pronounced heat stability and acid resistance of any of the six known types of plant sweet proteins. However, mabinlin II is difficult to extract from the Capparis masaikai plant, which is itself becoming increasingly scarce. This limits the use of naturally acquired mabinlin II. In this study, recombinant mabinlin II proteins were expressed and purified in Escherichia coli and in food-grade Lactococcus lactis. Recombinant mabinlin II proteins MBL-BH (containing the B-chains of mabinlin II downstream fused with His-tag) and MBL-ABH (containing the A- and B-chains of mabinlin II downstream fused with His-tag) were expressed in E. coli in the form of inclusion bodies. They were then purified and renatured. The refolded MBL-BH was found to be 100 times sweeter than sucrose by weight, but it was not heat-stable. Refolded MBL-ABH was neither sweet nor heat-stable. Recombinant mabinlin II proteins were secreted and expressed intracellularly in food-grade L. lactis, in which the concentrated cell samples and culture medium samples were detected using enzyme-linked immunosorbent assay and Western blotting analysis with anti-mabinlin II polyclonal antibody. This study demonstrated that the single B chain of mabinlin II has a sweet taste. The recombinant mabinlin II proteins have been successfully expressed in food-grade L. lactis, which is a crucial step in the production of mabinlin II through microorganism expression systems.