An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.
An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.
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DOI：
10.1039/d2lc00517d
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发表时间：
2023-01-17
期刊：
影响因子：
6.1
通讯作者：
Chang, Hsueh-Chia
中科院分区：
文献类型：
--
作者：
McCarthy, Kyle P.;Go, David B.;Senapati, Satyajyoti;Chang, Hsueh-Chia
核糖核蛋白（Ribonucleoproteins，RNP），特别是microRNA诱导的沉默复合物（microRNA-induced silencing complex，miRISC），与肿瘤相关基因调控有关。miRISC复合物或let-7 lin 28 A复合物中发现的特定RNA-蛋白质缔合可以下调肿瘤抑制基因，并与癌症直接相关。高蛋白质-RNA静电结合亲和力对于相关microRNA（miRNAs）的定量是一个特别的挑战。我们在这里报告的第一个微流控即时检测，允许直接定量RNP相关的RNA，这有可能大大推进RNP分析液体活检。该技术的关键是集成的阳离子-阴离子交换膜（CEM/AEM）平台，用于RNP（Cas9-miR-21）复合物的快速和不可逆解离（k = 0.0025 s−1），并在40分钟内定量其相关的miR-21。使用CEM诱导的耗尽前沿将RNP浓缩在耗尽前沿，使得浓度边界层内的高电场（>100 V cm−1）诱导低KD（~0.5 nM）复合物的不可逆解离，即使缔合速率（kon = 6.1 s−1）高1000倍，解离率也为~100%。高场还将解离的RNA从浓缩区中分离出来，而不重新结合。Cy 3标记的miR-21的检测限为1.1 nM。
Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s−1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm−1) within the concentration boundary layer induces irreversible dissociation of the low KD (~0.5 nM) complex, with ~100% dissociation even though the association rate (kon = 6.1 s−1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.
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