Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA.
College of Biological Sciences, University of California, Davis, CA, 95616, USA.
Biosens Bioelectron. 2020 May 1;155:112100. doi: 10.1016/j.bios.2020.112100. Epub 2020 Feb 18.
Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.
用于下一代医疗保健的个性化医学的蓬勃发展突出了对疾病生物标志物进行定量的敏感和准确的即时检测平台的需求。广泛展示的成簇规律间隔短回文重复序列 (CRISPR) 作为一种准确的基因编辑工具也表明，CRISPR 系统的高特异性和可编程性可用于开发生物传感系统。在此，我们提出了一种具有空前灵敏度和准确性的 CRISPR Cas 系统增强型电化学 DNA (E-DNA) 传感器。E-DNA 传感器的原理是表面信号探针（包含电化学标签）的目标诱导构象变化，导致电化学标签的电子转移率发生变化。通过将 CRISPR 切割活性引入 E-DNA 传感器，可以实现目标存在与否时更明显的信号变化。我们比较了 Cas9 和 Cas12a 增强的 E-DNA 传感器的性能，并优化了 CRISPR 的化学环境，实现了无酶扩增的皮摩尔检测限。此外，我们将 CRISPR 切割信号与原始 E-DNA 信号相关联，作为指示目标序列中潜在错配的策略。与基于经典 DNA 电化学的突变检测策略相比，CRISPR 增强的 E-DNA 传感器可以在未知浓度条件下确定单个突变的存在。总的来说，我们相信 CRISPR 增强的 E-DNA 传感策略由于其可编程性、模块化、高灵敏度和高精度，将特别适用于即时检测系统。
