通过暗场成像和磁性金纳米粒子实现 CRISPR/Cas13a 辅助的无扩增 miRNA 生物传感器

IF 3.5 Q2 CHEMISTRY, ANALYTICAL
Jae-Jun Kim, Jae-Sang Hong, Hyunho Kim, Moonhyun Choi, Ursula Winter, Hakho Lee and Hyungsoon Im
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引用次数: 0

摘要

微RNA(miRNA)是一种短的(约18-24个核苷酸)非编码RNA,已成为包括癌症在内的各种疾病的潜在生物标记物。由于其长度较短,在传统的扩增方法中,特异性往往成为一个问题。下一代测序技术可以作为一种替代方法,但其分析时间长、成本高昂,不太适合常规临床诊断。因此,开发一种利用简单、经济的系统进行快速、选择性和准确 miRNA 检测的方法至关重要。在这项工作中,我们报告了一种基于 CRISPR/Cas13a 的 miRNA 生物传感技术,该技术采用了床旁暗场(DF)成像技术。我们利用磁性金纳米粒子(MGNPs)复合物作为信号探针,该复合物由 200 nm 大小的磁珠(MBs)和 60 nm 大小的金纳米粒子(AuNPs)通过 DNA 杂交连接而成。一旦 CRISPR/Cas13a 系统识别到目标 miRNA(miR-21-5p),激活的 Cas13a 就会裂解含有 RNA 序列的桥连接体,从而释放出 60 nm-AuNPs 并通过便携式 DF 成像系统进行检测和量化。将 CRISPR/Cas13a、MGNPs 和 DF 成像相结合,在 30 分钟内对 miR-21-5p 进行了无扩增检测,检测限为 500 阿托摩尔,具有单碱基特异性。CRISPR/Cas13a辅助的MGNP-DF检测方法利用简单的设备实现了对miRNA的快速、选择性和准确检测,从而为癌症诊断提供了潜在的应用前景。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

CRISPR/Cas13a-assisted amplification-free miRNA biosensor via dark-field imaging and magnetic gold nanoparticles†

CRISPR/Cas13a-assisted amplification-free miRNA biosensor via dark-field imaging and magnetic gold nanoparticles†

MicroRNAs (miRNAs) are short (about 18–24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, including cancers. Due to their short lengths, the specificity often becomes an issue in conventional amplification-based methods. Next-generation sequencing techniques could be an alternative, but the long analysis time and expensive costs make them less suitable for routine clinical diagnosis. Therefore, it is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. In this work, we report a CRISPR/Cas13a-based miRNA biosensing using point-of-care dark-field (DF) imaging. We utilized magnetic-gold nanoparticle (MGNPs) complexes as signal probes, which consist of 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) linked by DNA hybridization. Once the CRISPR/Cas13a system recognized the target miRNAs (miR-21-5p), the activated Cas13a cleaved the bridge linker containing RNA sequences, releasing 60 nm-AuNPs detected and quantified by a portable DF imaging system. The combination of CRISPR/Cas13a, MGNPs, and DF imaging demonstrated amplification-free detection of miR-21-5p within 30 min at a detection limit of 500 attomoles (25 pM) and with single-base specificity. The CRISPR/Cas13a-assisted MGNP-DF assay achieved rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis.

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