Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States.
Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado 80523-1019, United States.
Anal Chem. 2024 Mar 12;96(10):4111-4119. doi: 10.1021/acs.analchem.3c04977. Epub 2024 Feb 28.
Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.
与单分析物检测相比，医学诊断中的多重分析被广泛认为是一种更全面、更彻底的方法。虽然聚合酶链反应和酶联免疫吸附测定（ELISA）等分析方法可用于生物标志物的多重检测，但这些方法仍然耗时且昂贵。侧向流动分析（LFA）是即时检测的一种有吸引力的选择，并且存在多重化的 LFA 示例。然而，这些设备受到测试线空间分辨率、大样本量需求、交叉反应和灵敏度差的限制。最近的工作已经开发出毛细管流动微流控 ELISA 平台作为 LFA 的更敏感替代品；然而，这些类型的设备上的多重检测尚未得到证明。在最初的 SARS-CoV-2 大流行之后，对快速、敏感的即时检测设备的需求变得更加明显。展望未来，能够区分具有相似表现症状的疾病的设备将是理想的家庭诊断。在这里，首次报道了用于同时检测多种生物标志物的多重毛细管流动免疫分析装置的示例。从单个样品添加步骤开始，就可以将两种同时进行的 ELISA 所需的试剂和洗涤步骤输送到空间分离的测试条上。可以在 <15 分钟内获得视觉结果，并且可以使用智能手机捕获的图像进行定量数据分析。该设备用于区分和定量甲型 H1N1 血凝素（HA）和 SARS-CoV-2 核衣壳蛋白（N-蛋白）。使用该设备，分别获得了血凝素和核衣壳蛋白的分析检测限为 840 和 133 pg/mL。设备中存在一个靶标不会增加另一个测试线上的信号，表明两种测定之间没有交叉反应。此外，还同时检测了 N-蛋白和 HA，以及同时检测了 N-蛋白和人 C-反应蛋白（CRP）。已证明 SARS-CoV-2 感染患者中 CRP 水平升高与更严重的结果和更高的死亡风险相关。为了进一步扩展同时检测两种生物标志物的功能，同时检测了 CRP 和 N-蛋白，并且当两个目标都存在于样本中时，SARS-CoV-2 N-蛋白的存在不会干扰 CRP 的检测。
