Colorimetric detection of alkaline phosphatase based on the off-on effect of light-responsive oxidase mimicking activity of covalent organic framework (Cu-TpBpy-COF) under near-neutral condition.

A colorimetric strategy has been developed for the detection of alkaline phosphatase (ALP) activity based on the off-on effect of the catalytic activity of light-responsive oxidase mimics covalent organic framework (Cu-TpBpy-COF) in near-neutral condition. Cu-TpBpy-COF can effectively catalyze the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (oxTMB) with an absorption peak at 652 nm. Cu2+ is the active center of Cu-TpBpy-COF and pyrophosphate (PPi) can form a complex with Cu2+ to weaken the catalytic activity of Cu-TpBpy-COF. In the presence of ALP, PPi is hydrolyzed into orthophosphates (Pi) with low affinity to Cu2+, thus resulting in absorbance restoration. The absorbance at 652 nm is related to ALP activity in the linear range 10-150 U·L-1 with a detection limit of 7.17 U·L-1. The recoveries of ALP in serum samples are in the range 94.7~107.0% with relative standard deviations (RSD) lower than 5%. The decisive role of Cu2+ on the enhancing catalytic activities of Cu-TpBpy-COF in neutral condition was verified by TpBpy-COF and TpBD-COF as controls, in which the main difference between them is that TpBpy-COF contains pyridine nitrogen. Upon Cu2+ modification, Cu-TpBpy-COF has better catalytic activity than TpBpy-COF in a broader pH range because of the in situ generation of Cu+ under irradiation.

Generation of 3'-OH terminal-triggered encoding of multicolor fluorescence for simultaneous detection of different DNA glycosylases.

Uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG) are the important DNA glycosylases for initiating the repair of DNA damage, and the aberrant expression of DNA glycosylases is closely associated with various diseases, such as Parkinson's disease, several cancers, and human immunodeficiency. The simultaneous detection of UDG and hAAG is helpful for the study of early clinical diagnosis. However, the reported methods for multiple DNA glycosylase assay suffer from the application of an expensive single-molecule instrument, labor-tedious magnetic separation, and complicated design. Herein, we develop a simple fluorescence method with only three necessary DNA strands for the selective and sensitive detection of multiple DNA glycosylase activity based on the generation of 3'-OH terminal-triggered encoding of multicolor fluorescence. The method can achieve the detection limits of 5.5 × 10-5 U/mL for UDG and 3.3 × 10-3 U/mL for hAAG, which are lower than those of the reported fluorescence methods. Moreover, it can be further used to detect multiple DNA glycosylases in the human cervical carcinoma cell line (HeLa cells), normal human renal epithelial cells (293 T cells), and biological fluid and measure the enzyme kinetic parameters of UDG and hAAG.

Molecular Mechanism Associated With the Impact of Methane/Oxygen Gas Supply Ratios on Cell Growth of Methylomicrobium buryatense 5GB1 Through RNA-Seq.

The methane (CH4)/oxygen (O2) gas supply ratios significantly affect the cell growth and metabolic pathways of aerobic obligate methanotrophs. However, few studies have explored the CH4/O2 ratios of the inlet gas, especially for the CH4 concentrations within the explosion range (5∼15% of CH4 in air). This study thoroughly investigated the molecular mechanisms associated with the impact of different CH4/O2 ratios on cell growth of a model type I methanotroph Methylomicrobium buryatense 5GB1 cultured at five different CH4/O2 supply molar ratios from 0.28 to 5.24, corresponding to CH4 content in gas mixture from 5% to 50%, using RNA-Seq transcriptomics approach. In the batch cultivation, the highest growth rate of 0.287 h-1 was achieved when the CH4/O2 supply molar ratio was 0.93 (15% CH4 in air), and it is crucial to keep the availability of carbon and oxygen levels balanced for optimal growth. At this ratio, genes related to methane metabolism, phosphate uptake system, and nitrogen fixation were significantly upregulated. The results indicated that the optimal CH4/O2 ratio prompted cell growth by increasing genes involved in metabolic pathways of carbon, nitrogen and phosphate utilization in M. buryatense 5GB1. Our findings provided an effective gas supply strategy for methanotrophs, which could enhance the production of key intermediates and enzymes to improve the performance of bioconversion processes using CH4 as the only carbon and energy source. This research also helps identify genes associated with the optimal CH4/O2 ratio for balancing energy metabolism and carbon flux, which could be candidate targets for future metabolic engineering practice.