反式激活crRNA
化学
清脆的
核酸
滚动圆复制
环介导等温扩增
自愈水凝胶
计算生物学
纳斯巴
纳米技术
微流控
核糖核酸
DNA
生物化学
基因组编辑
基因
生物
DNA复制
有机化学
材料科学
作者
Rujian Zhao,Yidan Tang,Defeng Song,Meng Liu,Bingling Li
标识
DOI:10.1021/acs.analchem.3c03900
摘要
Recent advances have demonstrated the significant potential and advantages to repurpose existing point-of-care reactions/devices to realize portable detection of nonoriginal targets, e.g., pathogen genes. However, pursuing this aim usually requires protein indicator–nucleic acid conjugation via a covalent bond, which may bring drawbacks such as high cost, complicated procedure, and annoying component rebuilding. Herein, we developed a conjugation-free, effective, and universal detection platform called CRIs-gel (CRISPR/Cas12a-Responsive Indicators@RCA hydrogels). Various protein indicators are pre-encapsulated into the hydrogels made of effective and high-yield rolling circle amplification (RCA). Upon a targeting sequence binding with its antisense crRNA, CRISPR/Cas12a starts its trans-cleavage activity to crush the hydrogel, which may directly release the indicator for downstream readout. Two proteins, amylase (GA) and human chorionic gonadotropin (hCG), are successfully used as model indicators to trigger the downstream amylum-I2 color change and pregnancy test strip response. After coupling with upstream isothermal nucleic acid amplification, both portable readouts may detect as few as 2 copies/μL genetic sequences of influenza A virus (FluA), human papilloma virus (HPV), SARS-CoV-2, and influenza B virus (FluB). This conjugation-free CRIs-gel platform is thus simple, sensitive, and universal and can provide innovative insights for portable point-of-care testing (POCT) development.
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