SRI International, Menlo Park, CA, USA.
Clin Chem. 2013 Apr;59(4):641-8. doi: 10.1373/clinchem.2012.200360. Epub 2013 Jan 30.
Lateral flow assays (LFAs) are popular point-of-care diagnostic tools because they are rapid and easy to use. Nevertheless, they often lack analytical sensitivity and quantitative output and may be difficult to multiplex, limiting their usefulness in biomarker measurement. As a proof-of-concept study, we detail the design of a quantitative, multiplex LFA with readily available near-infrared (NIR) detection to improve analytical sensitivity.
NIR dye was conjugated to selected antibodies and incorporated into LFAs. We used singleplex, optimized NIR-LFAs to measure interleukin (IL)-6 from 0 to 200 pg/mL and developed duplex assays to simultaneously measure IL-6 from 0 to 100 pg/mL (0 to 4.5 pmol/L) and C-reactive protein (CRP) from 50 to 2500 ng/mL (0.4 to 20 nmol/L) on a single test strip. Assays were tested on 60 different spiked samples and compared to ELISA results.
NIR-LFAs detected IL-6 in a 10% plasma matrix with a limit of detection of 4 pg/mL (182 fmol/L) and a CV <7%. Duplex NIR-LFAs quantitatively measured IL-6 and CRP concentrations simultaneously. Values strongly correlated to ELISA measurements, with R(2) values of 0.9825 and 0.9711 for IL-6 and CRP, respectively.
NIR-LFAs exhibit quantitative measurement at pg/mL concentrations owing to a high signal-to-background ratio and robust detection antibody clearance through the test strip. Moreover, NIR-LFAs are able to detect molecules present at vastly different concentrations in multiplex format and compare favorably to ELISAs. LFAs with direct NIR detection may be a valuable tool for biomarker evaluation in the point-of-care setting.
侧向流动检测（LFA）作为一种即时诊断工具，因其快速、易用而广受欢迎。然而，它们通常缺乏分析灵敏度和定量输出，且难以实现多重检测，这限制了它们在生物标志物测量中的应用。作为概念验证研究，我们详细设计了一种具有定量、多重检测能力的近红外（NIR）检测侧向流动检测，以提高分析灵敏度。
将 NIR 染料与选定的抗体结合并掺入 LFAs 中。我们使用单重、优化的 NIR-LFA 来测量 0 至 200pg/mL 的白细胞介素（IL）-6，并开发了双重检测方法，以同时测量 0 至 100pg/mL（0 至 4.5pmol/L）的 IL-6 和 50 至 2500ng/mL（0.4 至 20nmol/L）的 C 反应蛋白（CRP）。在 60 个不同的加标样本中对检测进行了测试，并与 ELISA 结果进行了比较。
NIR-LFA 在 10%血浆基质中检测 IL-6 的检测限为 4pg/mL（182fmol/L），CV<7%。双重 NIR-LFA 能够同时定量测量 IL-6 和 CRP 浓度。与 ELISA 测量值具有很强的相关性，IL-6 和 CRP 的 R2 值分别为 0.9825 和 0.9711。
由于高信噪比和通过测试条清除检测抗体，NIR-LFA 可以在 pg/mL 浓度下进行定量测量。此外，NIR-LFA 能够以多重检测格式检测存在于差异极大浓度的分子，并与 ELISA 相比具有优势。具有直接 NIR 检测的 LFAs 可能是即时护理环境中生物标志物评估的有价值工具。
