Multiplexing cytokine analysis: towards reducing sample volume needs in clinical diagnostics
Multiplexing cytokine analysis: towards reducing sample volume needs in clinical diagnostics
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DOI：
10.1039/c9an00297a
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发表时间：
2019-05-21
期刊：
影响因子：
4.2
通讯作者：
Daunert，Sylvia
中科院分区：
文献类型：
--
作者：
Yu，Xiaowen;Scott，Daniel;Daunert，Sylvia
改善更精确的疾病诊断和管理的趋势在很大程度上依赖于对患者生理样本中生物标志物的测量。理想情况下，最终目标是在一滴血中检测尽可能多的临床相关生物标志物，在小体积生理样本中实现快速、敏感、可重复和负担得起的检测。生物发光（BL）蛋白提供了这种标记所需的许多特性，包括在极低浓度下检测，不受生理液体的干扰，导致极好的检测限，以及与许多小型化系统的兼容性。然而，迄今为止，BL蛋白的使用受到其有限的多路复用能力的限制。BL蛋白通常表现出单一的发射谱图和衰变动力学，使得同时检测多种分析物变得困难。该领域的最新进展包括使用两种不同的工程发光蛋白通过一维时间分辨率来实现分辨信号。然而，到目前为止，这种方法只导致双重分析物检测。在此，我们已经证明，使用结合时间和空间分辨率的二维方法，我们可以扩展生物发光蛋白的多路复用能力。为此，光蛋白aequorin （AEQ）被用于在单孔中同时检测三种不同的分析物，通过使用三个离散的时间/波长窗口进行区分。通过位点特异性突变和合成腔肠嘧啶的结合，“半合成”AEQ变体已经被开发出来，具有改变的发射谱和衰变动力学。在这项研究中，两种AEQ突变蛋白与三种促炎细胞因子（肿瘤坏死因子α、白细胞介素6和8）基因结合，产生AEQ标记的细胞因子。这些融合蛋白与合成腔肠嘧啶结合，产生具有不同发射最大值和半衰期的蛋白质，从而允许在单个样品中同时检测所有三种细胞因子。通过使用人体生理样本，并将我们的结果与市售的三种细胞因子的单独测试进行比较，在血清中证明了该测定的有效性。
The trend for improved more precise diagnostics and management of disease heavily relies on the measurement of panels of biomarkers in physiological samples of patients. Ideally, the ultimate goal would be to detect as many clinically relevant biomarkers as possible in a single drop of blood, achieving quick, sensitive, reproducible, and affordable detection in small volume physiological samples. Bioluminescent (BL) proteins provide many of the desired characteristics required for such labels, including detection at extremely low concentrations, no interference from physiological fluids leading to excellent detection limits, and compatibility with many miniaturized systems. However, to date the use of BL proteins has been restricted by their limited multiplexing capabilities. BL proteins typically exhibit a single emission profile and decay kinetics making the simultaneous detection of multiple analytes difficult. Recent progresses in this area include the use of two different engineered luminescent proteins to achieve resolved signals via one-dimensional time resolution. This approach, however, to date only lead to a dual analyte detection. Herein, we have demonstrated that using a two-dimensional approach that combines both temporal and spatial resolution, we can expand the multiplexing capabilities of bioluminescent proteins. To that end, the photoprotein aequorin (AEQ) has been employed for the simultaneous detection of three separate analytes in a single well, differentiated through the use of three discrete time/wavelength windows. Through a combination of site-specific mutations and synthetic coelenterazines “semi-synthetic” AEQ variants have been developed with altered emission profiles and decay kinetics. In this study, two AEQ mutant proteins were genetically conjugated to three pro-inflammatory cytokines (tumor necrosis factor alpha, interleukins 6 and 8) resulting in AEQ-labeled cytokines. These fusion proteins were combined with synthetic coelenterazines resulting in proteins with differing emission maxima and half-lives to allow for the simultaneous detection of all three cytokines in a single sample. The validity of the assay was demonstrated in serum by employing human physiological samples and comparing our results with commercially available individual tests for each of the three cytokines.