CRISPR Cas12a-Powered Silicon Surface-Enhanced Raman Spectroscopy Ratiometric Chip for Sensitive and Reliable Quantification,Analytical Chemistry

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CRISPR Cas12a-Powered Silicon Surface-Enhanced Raman Spectroscopy Ratiometric Chip for Sensitive and Reliable Quantification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-01-19 , DOI: 10.1021/acs.analchem.2c03990
Haiting Cao ₁ , Jingxuan Xie ₁ , Jiayi Cheng ₁ , Yanan Xu ₁ , Xing Lu ₁ , Jie Tang ₁ , Xiaojie Zhang ₂ , Houyu Wang ₁

Affiliation

Sensitive and reliable clustered regularly interspaced short palindromic repeats (CRISPR) quantification without preamplification of the sample remains a challenge. Herein, we report a CRISPR Cas12a-powered silicon surface-enhanced Raman spectroscopy (SERS) ratiometric chip for sensitive and reliable quantification. As a proof-of-concept application, we select the platelet-derived growth factor-BB (PDGF-BB) as the target. We first develop a microfluidic synthetic strategy to prepare homogeneous silicon SERS substrates, in which uniform silver nanoparticles (AgNPs) are in situ grown on a silicon wafer (AgNPs@Si) by microfluidic galvanic deposition reactions. Next, one 5′-SH-3′-ROX-labeled single-stranded DNA (ssDNA) is modified on AgNPs via Ag–S bonds. In our design, such ssDNA has two fragments: one fragment hybridizes to its complementary DNA (5′-Cy3-labeled ssDNA) to form double-stranded DNA (dsDNA) and the other fragment labeled with 6′-carboxy-X-rhodmine (ROX) extends out as a substrate for Cas12a. The cleavage of the ROX-tagged fragment by Cas12a is controlled by the presence or not of PDGF-BB. Meanwhile, Cy3 molecules serving as internal standard molecules still stay at the end of the rigid dsDNA, and their signals remain constant. Thereby, the ratio of ROX signal intensity to Cy3 intensity can be employed for the reliable quantification of PDGF-BB concentration. The developed chip features an ultrahigh sensitivity (e.g., the limit of detection is as low as 3.2 pM, approximately 50 times more sensitive than the fluorescence counterpart) and good reproducibility (e.g., the relative standard deviation is less than 5%) in the detection of PDGF-BB.

中文翻译：

CRISPR Cas12a 供电的硅表面增强拉曼光谱比例芯片，用于灵敏可靠的量化

在不对样本进行预放大的情况下进行灵敏可靠的成簇规则间隔短回文重复序列 (CRISPR) 量化仍然是一个挑战。在此，我们报告了一种由 CRISPR Cas12a 供电的硅表面增强拉曼光谱 (SERS) 比例芯片，用于灵敏和可靠的量化。作为概念验证应用，我们选择血小板衍生生长因子-BB (PDGF-BB) 作为目标。我们首先开发了一种微流控合成策略来制备均质硅 SERS 基板，其中通过微流控电沉积反应在硅晶片 (AgNPs@Si) 上原位生长均匀的银纳米粒子 (AgNPs)。接下来，一个 5'-SH-3'-ROX 标记的单链 DNA (ssDNA) 在 AgNPs 上被修饰银硫键。在我们的设计中，这样的 ssDNA 有两个片段：一个片段与其互补 DNA（5'-Cy3 标记的 ssDNA）杂交形成双链 DNA（dsDNA），另一个片段用 6'-羧基-X-罗丹明标记（ ROX) 作为 Cas12a 的底物延伸出去。Cas12a 对 ROX 标记片段的切割受控于 PDGF-BB 的存在与否。同时，作为内标分子的Cy3分子仍然停留在刚性dsDNA的末端，其信号保持不变。因此，ROX 信号强度与 Cy3 强度的比值可用于 PDGF-BB 浓度的可靠量化。开发的芯片具有超高灵敏度（例如，检测限低至 3.2 pM，比荧光对应物灵敏约 50 倍）和良好的重现性（例如，

更新日期：2023-01-19

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