Egloff Sylvie, Melnychuk Nina, Reisch Andreas, Martin Sophie, Klymchenko Andrey S
Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route Du Rhin, 67401, Illkirch, France.
Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route Du Rhin, 67401, Illkirch, France.
Biosens Bioelectron. 2021 May 1;179:113084. doi: 10.1016/j.bios.2021.113084. Epub 2021 Feb 10.
Detection of cellular microRNA biomarkers is an emerging powerful tool in cancer diagnostics. Currently, it requires multistep tedious protocols based on molecular amplification of the RNA target, e.g. RT-qPCR. Here, we developed a one-step enzyme-free method for microRNA detection in cellular extracts based on light-harvesting nanoparticle (nanoantenna) biosensors. They amplify the fluorescence signal by effective Förster resonance energy transfer (FRET) from ultrabright dye-loaded polymeric nanoparticle to a single acceptor and thus convert recognition of one microRNA copy (through nucleic acid strand displacement) into a response of >400 dyes. The developed nanoprobes of 17-19 nm diameter for four microRNAs (miR-21, let-7f, miR-222 and miR-30a) exhibit outstanding brightness (up to 3.8 × 10 Mcm) and ratiometric sequence-specific response to microRNA with the limit of detection (LOD) down to 1.3 pM (21 amol), equivalent to 24 RT-qPCR cycles. They enable quantitative detection of the four microRNAs in RNA extracts from five cancerous cell lines (human breast cancer - T47D and MCF7, head and neck cancer - CAL33 and glioblastoma - LNZ308 and U373) and two non-cancerous ones (Hek293 and MCF10A), in agreement with RT-qPCR. The results confirmed that let-7f and especially miR-21 are systematically overexpressed in all studied cancerous cell lines. These nanoparticle biosensors are compatible with low-cost portable fluorometers and small detection volumes (11 amol LOD), opening a route to rapid point-of-care cancer diagnostics.
细胞微小RNA生物标志物的检测是癌症诊断中一种新兴的强大工具。目前，它需要基于RNA靶标分子扩增的多步繁琐方案，例如逆转录定量聚合酶链反应（RT-qPCR）。在此，我们基于光捕获纳米颗粒（纳米天线）生物传感器开发了一种用于细胞提取物中微小RNA检测的一步无酶方法。它们通过从超亮染料负载的聚合物纳米颗粒到单个受体的有效荧光共振能量转移（FRET）来放大荧光信号，从而将一个微小RNA拷贝的识别（通过核酸链置换）转化为>400个染料的响应。所开发的针对四种微小RNA（miR-21、let-7f、miR-222和miR-30a）的直径为17-19nm的纳米探针表现出出色的亮度（高达3.8×10Mcm）和对微小RNA的比例序列特异性响应，检测限（LOD）低至1.3pM（21amol），相当于24个RT-qPCR循环。它们能够对来自五种癌细胞系（人乳腺癌 - T47D和MCF7、头颈癌 - CAL33以及胶质母细胞瘤 - LNZ308和U373）和两种非癌细胞系（Hek293和MCF10A）的RNA提取物中的四种微小RNA进行定量检测，结果与RT-qPCR一致。结果证实，let-7f尤其是miR-21在所有研究的癌细胞系中均系统性过表达。这些纳米颗粒生物传感器与低成本便携式荧光计和小检测体积（11amol LOD）兼容，为快速即时癌症诊断开辟了一条途径。
