NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China.
The Second Clinical Medical School, Southern Medical University, Guangzhou, 510515, China.
Anal Chim Acta. 2022 Aug 15;1221:340131. doi: 10.1016/j.aca.2022.340131. Epub 2022 Jun 28.
Elevated C-reactive protein (CRP) levels are linked with bacterial infection, local inflammation in osteoarthritis and the increased risk of developing cardiovascular disease. Here, a sensitive and label-free CRP assay is developed by combining cyclic enzymatic signal amplification and capillary electrophoresis (CE) with UV detection. This assay is constructed of base pairing and target recognition. Thereinto, nicking endonuclease (NEase) can recognize the specific nucleotide sequences in double-stranded DNA (dsDNA), which is formed by a CRP aptamer and its complementary DNA (cDNA). Sequentially, NEase cleaves only cDNA to produce signal DNAs. Therefore, a large number of signal DNAs are generated through continuous enzyme cleavage. In the presence of CRP, the aptamer recognizes and binds to CRP with high affinity and selectivity, which results in a decrease in signal DNAs, and thus the UV absorption value of CE significantly decreases, too. A wide linear range was obtained between 0.0125 and 15 μg mL (0.11-130.5 nM) in 1% human serum with a detection limit of 4 ng mL (35 pM). Additionally, the proposed method is universal and can be applied to analyze other similar substances by altering the matched aptamer.
C-反应蛋白（CRP）水平升高与细菌感染、骨关节炎局部炎症以及心血管疾病风险增加有关。在这里，我们结合循环酶信号放大和毛细管电泳（CE）与紫外检测，开发了一种灵敏且无标记的 CRP 分析方法。该方法基于碱基配对和目标识别。其中，切口酶（NEase）可以识别双链 DNA（dsDNA）中的特定核苷酸序列，该序列由 CRP 适体与其互补 DNA（cDNA）形成。随后，NEase 仅切割 cDNA 以产生信号 DNA。因此，通过连续酶切可产生大量的信号 DNA。在 CRP 存在的情况下，适体与 CRP 以高亲和力和选择性识别和结合，导致信号 DNA 减少，从而使 CE 的紫外吸收值也显著降低。在 1%人血清中，该方法在 0.0125 至 15 μg mL（0.11 至 130.5 nM）范围内具有较宽的线性范围，检测限为 4 ng mL（35 pM）。此外，该方法具有通用性，通过改变匹配的适体，可用于分析其他类似物质。
