Xueping Yang,Jingyun Li,Suixin Zhang,Chao Li,Jiehua Ma
CRISPR/Cas-based systems are highly attractive for developing next-generation diagnostic technologies because of their intrinsic merits such as simplicity, sensitivity, and specificity. However, currently, nucleic acid amplification procedures are still needed to achieve attomolar sensitivity in most CRISPR/Cas-based assays, which causes high cost, operation difficulty, and low efficiency. Herein, we combine the CRISPR/Cas12a-based assay and a single-microbead detection platform for one-step and amplification-free detection of DNA at the single-molecule level. By modifying DNA reporters on a biomimetic membrane-coated microbead, the activated Cas12a by targets will cleave these reporters and lighten the bead within 10 min. The method allows the detection of the target down to three copies in a 5 μL sample. Furthermore, we successfully apply this method for the specific identification of viral infection, foodborne bacteria, and DNA mutation in real samples without extra nucleic acid amplification. We believe that this approach offers new insights for developing CRISPR/Cas-based DNA assays in biomedical applications.
Lei Chao,Fan Wenjiao,Ren Wei,Duan Xinrui,Liu Chenghui
We propose a novel single microbead “chemiluminescence (CL) super enriching and imaging” strategy that efficiently concentrates highly intense, long-lasting, and ultrastable CL shining on only a single microbead (SMB), allowing for the ultrasensitive quantification of various biomarkers at the attomolar level. Aided by a phenothiazine derivative enhancer, the traditional flash-type CL of horseradish peroxidase (HRP)-H2O2-luminol can be efficiently converted into a significantly enhanced (103-fold), persistent, and ultrastable CL emission (E-CL) that can be exactly confined and accurately imaged on the microbead interface. Then, by employing only one minuscule-sized SMB as the sole reaction and signaling unit, the target as well as the aroused E-CL signal concentrated on the SMB can further achieve ~104 “super-enriching” efficiency. Therefore, by “super enriching” target-responsive E-CL signal on an SMB, the SMB E-CL imaging strategy provides a powerful tool for the attomolar-level detection of various biomarkers including proteins, microRNAs, and virus DNA.
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