背景:SARS-CoV-2感染的早期诊断对于控制COVID-19爆发仍然至关重要。传统的聚合酶链反应,酶联免疫吸附测定或侧流免疫测定在检测时间上表现不佳,样品制备过程和准确性。基于表面增强拉曼散射(SERS)的检测已成为一种强大的分析技术,克服了上述限制。然而,由于传统衬底的近场效应,难以监测适体与蛋白质的结合事件。很明显,需要一种支持扩展和更强电磁场的新型SERS基底来保持长程效应并允许结合事件测试。
结果:在这一挑战的推动下,我们报道了一种远程SERS活性底物,它是通过在折射率对称的环境和Au反射镜表面中插入领结纳米孔径阵列而构建的,用于SARS-CoV-2蛋白结合事件检测。然后,双π结构aptasensor是通过spike(S)和核衣壳(N)蛋白适体的杂交简单地设计的,和相应的互补链。这种双π结构会在靶蛋白S和N存在时解离,导致SERS反应降低,奠定了我们系统的检测基础。更重要的是,由于涉及两个拉曼标记,蛋白质S和N可以同时被感知。我们提出的方法在较宽的浓度范围内显示出提高的灵敏度和较低的多重检测限(蛋白质S为1.6×10-16g/mL,蛋白质N为1.0×10-16g/mL)。
结论:这代表了用于同时检测S和N蛋白的第一个远程SERSapatasensor平台。我们的方法显示出高灵敏度,选择性,再现性,唾液和血清样品中的稳定性和显着回收率,这对于COVID的早期诊断以及未来未知的冠状病毒尤为重要。
BACKGROUND: Early diagnosis of SARS-CoV-2 infection is still critical to control COVID-19 outbreak. Traditional polymerase chain reaction, enzyme-linked immunosorbent assay or lateral flow immunoassay performed poorly on detection times, sample preparation process and accuracy. Surface-enhanced Raman scattering (SERS)-based detection has emerged as a powerful analytical technique, which overcomes the above limitations. However, due to the near-field effect of traditional substrate, it is difficult to monitor the binding event of aptamers with proteins. It is obvious that a novel SERS substrate thatsupportedextended and stronger electromagnetic fields was required to hold long-range effects and allow for binding event testing.
RESULTS: Driven by this challenge, we reported a long-range SERS-active substrate, which was built by inserting bowtie nanoaperture arrays in a refractive-index-symmetric environment and Au mirror surfaces, for SARS-CoV-2 protein binding event detection. Then, a double-π structure aptasensor was simply designed through the hybridization of spike (S) and
nucleocapsid (N) proteins aptamers, and a corresponding complementary strand. This kind of double-π structure would dissociate when targets proteins S and N existed and led to the SERS responses decreased, which established the detection basis of our system. What\'s more, due to two Raman labels were involved, both proteins S and N can be sensed simultaneously. Our proposed method showed improved sensitivity with a low limit of detection for multiplex detection (1.6 × 10-16 g/mL for protein S and 1.0 × 10-16 g/mL for protein N) over a wide concentration range.
CONCLUSIONS: This represents the first long-range SERS apatasensor platform for detection of S and N proteins simultaneously. Our method showed high sensitivity, selectivity, reproducibility, stability and remarkable recoveries in human in saliva and serum samples, which is particularly important for the early diagnostics of COVID as well as for future unknown coronavirus.