Discrimination of Chinese green tea according to tea polyphenols using fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks.

A facile and rapid fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks was proposed for Chinese green tea discrimination. According to large porosity of Tb@UiO-66-(COOH)2 and Eu@UiO-66-(COOH)2, phenolic hydroxyl groups of tea polyphenols could coordinate with free carboxylic acid groups and was captured into the pores, which led to the disturbance of electronic structure of ligand and inhibited the energy transfer efficiency from ligand to Tb (III) and Eu (III) center, causing the fluorescence quenching effect. Based on Hierarchy Cluster Analysis and Linear Discrimination Analysis, the fluorescence sensor array was employed for successful tea polyphenols classification through the analysis of different fluorescence quenching effect to tea polyphenols. Green tea samples within different categories and grades were also successfully discriminated using this assay according to tea polyphenols, providing a new method for Chinese green tea identification.

Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection.

High-performance detection of DNA methylation possesses great significance for the diagnosis and therapy of cancer. Herein, for the first time, we present a digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system (DESCS) for accurate and sensitive determination of site-specific DNA methylation. This dual methylation-sensitive restriction endonuclease system selectively digests the unmethylated target but exhibits no response to methylated DNA. Therefore, the intact methylated DNA target triggers the RPA reaction for rapid signal amplification. In contrast, the digested unmethylated target initiates no RPA reaction. RPA products with a T7 promoter can execute the T7 transcription in the presence of T7 RNA polymerase to generate a large number of single-stranded RNA (ssRNA). This ssRNA can be recognized by CRISPR/Cas13a to induce the ssRNase activity of Cas13a, showing the indiscriminate cleavage of the collateral FQ reporter to release the fluorescence signal. With such a design, by combining the unique features of dual methylation-sensitive restriction endonucleases with RPA-assisted CRISPR/Cas13a, the DESCS system not only presents the rapid and powerful signal amplification for the determination of methylated DNA with ultrahigh sensitivity but also effectively eliminates the false positive influences from incomplete digestion of the unmethylated target. More importantly, 0.01% methylation level can be effectively distinguished with the existence of excess unmethylated DNA. In addition, the DESCS assay is integrated into the lateral flow biosensor (LFB) for the point-of-care determination of DNA methylation. In view of the superiorities in high sensitivity, outstanding selectivity, and ease of operation, the DESCS system will provide a reliable assay for site-specific analysis of methylation.