Sampieri Alicia, Monroy-Contreras Ricardo, Asanov Alexander, Vaca Luis
Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular, UNAM, Ciudad Universitaria, Mexico, Mexico.
TIRF Labs, Cary, NC, United States.
Front Bioeng Biotechnol. 2022 Jul 22;10:881679. doi: 10.3389/fbioe.2022.881679. eCollection 2022.
We have developed a novel microarray system based on three technologies: 1) molecular beacons designed to interact with DNA targets at room temperature (25-27°C), 2) tridimensional silk-based microarrays containing the molecular beacons immersed in the silk hydrogel, and 3) shallow angle illumination, which uses separated optical pathways for excitation and emission. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing, and stringency control, and measure only end-point results, our microarray technology provides enhanced signal-to-background ratio (achieved by separating the optical pathways for excitation and emission, resulting in reduced stray light), performs analysis rapidly in one step without the need for labeling DNA targets, and measures the entire course of association kinetics between target DNA and the molecular beacons. To illustrate the benefits of our technology, we conducted microarray assays designed for the identification of influenza viruses. We show that in a single microarray slide, we can identify the virus subtype according to the molecular beacons designed for hemagglutinin (H1, H2, and H3) and neuraminidase (N1, N2). We also show the identification of human and swine influenza using sequence-specific molecular beacons. This microarray technology can be easily implemented for reagentless point-of-care diagnostics of several contagious diseases, including coronavirus variants responsible for the current pandemic.
1）设计用于在室温（25 - 27°C）下与DNA靶标相互作用的分子信标；2）包含浸入丝水凝胶中的分子信标的三维丝基微阵列；3）浅角照明，其使用分开的光路进行激发和发射。与传统微阵列不同，传统微阵列的信噪比降低，需要几个孵育、冲洗和严格控制的阶段，并且仅测量终点结果，我们的微阵列技术提供了更高的信噪比（通过分离激发和发射的光路实现，减少了杂散光），无需对DNA靶标进行标记即可一步快速进行分析，并测量靶标DNA与分子信标之间结合动力学的整个过程。为了说明我们技术的优势，我们进行了旨在鉴定流感病毒的微阵列分析。我们表明，在单个微阵列载玻片上，我们可以根据针对血凝素（H1、H2和H3）和神经氨酸酶（N1、N2）设计的分子信标来鉴定病毒亚型。我们还展示了使用序列特异性分子信标对人类和猪流感的鉴定。这种微阵列技术可以很容易地用于几种传染病的无需试剂的即时诊断，包括导致当前大流行的冠状病毒变种。
