Lele Yang,Sijia Wu,Jinchao Wei,Jiagang Deng,Xiaotao Hou,Erwei Hao,Lei Zhang,Peng Li
Glucose is a primary source of energy used in most organisms. Thus, development of reliable approaches to measure intracellular glucose uptake is an important research issue. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy- d -glucose (2-NBDG), as a fluorescent glucose derivative, has been widely used to track intracellular glucose uptake by fluorescence imaging and measuring in mammalian cells. However, the avoid-less cross-interference of intrinsic autofluorescence background and tested fluorescent compounds limits its ability to provide trustworthy information on intracellular glucose uptake. By the extraction, separation and detection of 2-NBDG, a simple, sensitive and accurate HPLC-FLD method was established and validated for the measurement of intracellular glucose uptake in HepG2 cells. The developed method has been employed successfully to assess the glucose uptake activity of anti-diabetic drugs and fluorescent natural products. A fit-for-purpose partial validation was further performed for quantification and comparison of glucose uptake in AML12, LO2 hepatocytes, L6 myoblasts and 3T3-L1 preadipocytes.
This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.
危险品化学品经营许可证（带存储）