Chenchen Ge,Juan Feng,Jiaming Zhang,Kai Hu,Dou Wang,Ling Zha,Xuejuan Hu,Rongsong Li
Nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV2 and is highly conserved, and there are no homologous proteins in the human body, making it an ideal biomarker for the early diagnosis of SARS-CoV2. However, early detection of clinical specimens for SARS-CoV2 remains a challenge due to false-negative results with viral RNA and host antibodies based testing. In this manuscript, a microfluidic chip with femtoliter-sized wells was fabricated for the sensitive digital detection of N protein. Briefly, β-galactosidase (β-Gal)-linked antibody/N protein/aptamer immunocomplexes were formed on magnetic beads (MBs). Afterwards, the MBs and β-Gal substrate fluorescein-di-β- d -galactopyranoside (FDG) were injected into the chip together. Each well of the chip would only hold one MB as confined by the diameter of the wells. The MBs in the wells were sealed by fluorocarbon oil, which confines the fluorescent (FL) product generated from the reaction between β-Gal and FDG in the individual femtoliter-sized well and creates a locally high concentration of the FL product. The FL images of the wells were acquired using a conventional inverted FL microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA).
Lifei Gu,Xueqing Xie,Bing Wang,Yibao Jin,Lijun Wang,Guo Yin,Jue Wang,Kaishun Bi,Tiejie Wang
Lonicerae japonicae flos (L. japonicae flos, Lonicera japonica Thunb.) is one of the most commonly prescribed botanical drugs in the treatment or prevention of corona virus disease 2019. However, L. japonicae flos is often confused or adulterated with Lonicerae flos (L. flos, Lonicera macrantha (D.Don) Spreng., Shanyinhua in Chinese). The anti-SARS-CoV2 activity and related differentiation method of L. japonicae flos and L. flos have not been documented. In this study, we established a chemical pattern recognition model for quality analysis of L. japonicae flos and L. flos based on ultra-high performance liquid chromatography (UHPLC) and anti-SARS-CoV2 activity. Firstly, chemical data of 59 batches of L. japonicae flos and L. flos were obtained by UHPLC, and partial least squares-discriminant analysis was applied to extract the components that lead to classification. Next, anti-SARS-CoV2 activity was measured and bioactive components were acquired by spectrum-effect relationship analysis. Finally, characteristic components were explored by overlapping feature extracted components and bioactive components. Accordingly, eleven characteristic components were successfully selected, identified, quantified and could be recommended as quality control marker. In addition, chemical pattern recognition model based on these eleven components was established to effectively discriminate L. japonicae flos and L. flos. In sum, the demonstrated strategy provided effective and highly feasible tool for quality assessment of natural products, and offer reference for the quality standard setting.
危险品化学品经营许可证（带存储）