Hu Juan, Kim Joonyul, Easley Christopher J
Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, AL 36849.
Anal Methods. 2015 Sep 7;7(17):7358-7362. doi: 10.1039/c5ay00837a.
Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (-dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument-even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding.
有效的基于适配体的蛋白质检测需要与适配体 - 蛋白质结合的定量报告分子偶联。通常，这涉及对DNA杂交的直接光学或电化学读出，或与读出偶联的扩增步骤。然而，方法开发常常受到适配体 - 靶标结合机制多样性的阻碍，这些机制可能会干扰杂交步骤。作为一种更简单且更具通用性的适配体 - 蛋白质结合读出方法，我们报告热荧光分析（TFA）可用于定量检测蛋白质水平。以血小板衍生生长因子（PDGF）及其相应适配体的亚纳摩尔检测（0.74 nM）作为测试案例展示。在存在各种DNA嵌入染料的情况下，与嵌入的未结合适配体相比，与蛋白质结合的适配体表现出荧光强度的变化。这使得能够使用荧光熔解分析（-dF/dT曲线）对结合和未结合的适配体进行热分辨。值得注意的是，这种均相光学方法能够扣除人血清中的自发荧光，在1:7和1:3稀释的血清中，PDGF的检测限分别为1.8 nM和10.7 nM。我们由此证明，即使在人血清中，使用简单的qPCR仪器也能以均相形式对结合和未结合的适配体进行热分辨。这种方法的简单性为实现强大、通用的适配体 - 蛋白质结合读出迈出了重要一步。
