Immunoassays-Immunosensors Lab, Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR "Demokritos", 15341 Aghia Paraskevi, Greece.
Institute of Nanoscience and Nanotechnology, NCSR "Demokritos", 15341 Aghia Paraskevi, Greece.
Biosensors (Basel). 2023 Sep 1;13(9):865. doi: 10.3390/bios13090865.
The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications.
新冠疫情凸显了对适合快速可靠诊断病毒感染的即时检测方法的迫切需求。为满足这一需求，我们报告了一种基于多区域反射光谱学的生物分析设备，可快速、无标记地同时测定两种 SARS-CoV-2 蛋白，即核蛋白和 S1 蛋白的受体结合域肽。通过在顶部具有不同二氧化硅厚度的相邻区域的硅芯片来实现对这两种蛋白的同时检测，每个区域都修饰有一种针对核蛋白或 SARS-CoV-2 的受体结合域的抗体。单个探针同时照亮两个区域，并收集反射光，将其引导至光谱仪。通过将来自两个二氧化硅区域的组合反射光谱在线转换为各自的吸附层厚度，实现了实时监测两个区域上发生的免疫反应。已经测试了几种抗体来定义这种配对，在非竞争免疫测定格式下提供了更高的特异性信号。生物素化的二级抗体和链霉亲和素用于增强特异性信号。两种蛋白均在 12 分钟内检测到，检测限为 1.0ng/mL。该测定法具有较高的重复性，内和间测定变异系数均低于 10%。此外，从商业的用于从鼻咽拭子采集 SARS-CoV-2 的自测试试剂盒中提取缓冲液中制备的加标样品中，两种蛋白的回收率均在 90.0%至 110%之间。短的测定时间与出色的分析性能和紧凑的仪器尺寸相结合，使所提出的设备和测定法适用于即时检测应用。
