Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing
Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing
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用于现场核酸检测的超灵敏 CRISPR/Cas12a 驱动的 SERS 生物传感器及其在牛奶真伪检测中的应用
DOI：
10.1021/acs.jafc.1c08262
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发表时间：
2022
影响因子：
6.1
通讯作者：
Guoliang Li
中科院分区：
文献类型：
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作者：
Ruiyuan Pan;Jianghua Liu;Panxue Wang;Di Wu;Jian Chen;Yongning Wu;Guoliang Li
提出了一种由CRISPR/Cas12a驱动的超灵敏表面增强拉曼散射（SERS）生物传感器，用于现场核酸检测。我们巧妙地用靶反应普鲁士蓝（PB）纳米标签修饰单链DNA (ssDNA)，形成探针并将其固定在微孔板上。由于CRISPR/Cas12a效应体特异的碱基配对和高效的反式切割能力，可以分别实现精确的靶DNA识别和信号扩增。在目标DNA存在的情况下，指向探针的反式切割被激活，导致一定数量的PB纳米颗粒（NPs）的释放。然后，这些游离的PB NPs将被移除。碱处理触发微孔板中剩余PB NPs的分解，产生大量的铁氰化物阴离子（Fe(CN)64 -），在“生物拉曼沉默区”表现出独特的特征拉曼峰。通过将碱处理过的溶液与SERS底物Au@Ag核壳NP混合，最终将目标DNA的浓度显示为无干扰背景的SERS信号，可通过便携式拉曼光谱仪检测。重要的是，该策略可以显示对目标DNA的超低检测限为224 aM。此外，通过将牛奶作为羊奶中的掺假成分，该生物传感器成功应用于羊奶的真实性检测。
An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64–), which could exhibit a unique characteristic Raman peak that was located in the “biological Raman-silent region”. By mixing the alkali-treated solution with the SERS substrate, Au@Ag core–shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.