Label-free and highly sensitive detection of CRP based on the combination of nicking endonuclease-assisted signal amplification and capillary electrophoresis-UV assay
Label-free and highly sensitive detection of CRP based on the combination of nicking endonuclease-assisted signal amplification and capillary electrophoresis-UV assay
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DOI：
10.1016/j.aca.2022.340131
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发表时间：
2022-07-01
影响因子：
6.2
通讯作者：
Liu，Lihong
中科院分区：
文献类型：
--
作者：
Tang，Qing;Xu，Jun;Liu，Lihong
C反应蛋白（CRP）水平升高与细菌感染、骨关节炎局部炎症和心血管疾病风险增加有关。在这里，一个敏感的和无标记的CRP检测开发相结合的循环酶信号放大和毛细管电泳（CE）与紫外检测。该测定由碱基配对和靶识别构成。其中，切口核酸内切酶（nicking endonuclease，NEase）可识别CRP适体与其互补DNA（cDNA）形成的双链DNA（double-stranded DNA，dsDNA）中的特定核苷酸序列。顺序地，NEase仅切割cDNA以产生信号DNA。因此，通过连续的酶切割产生大量的信号DNA。在CRP存在的情况下，适体以高亲和力和选择性识别并结合CRP，这导致信号DNA减少，因此CE的UV吸收值也显著降低。在1%人血清中，线性范围为0.0125 - 15 μg mL−1（0.11-130.5 nM），检测限为4 ng mL−1（35 pM）。该方法具有通用性，可通过改变匹配的适体来分析其他类似物质。
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−1(0.11–130.5 nM) in 1% human serum with a detection limit of 4 ng mL−1(35 pM). Additionally, the proposed method is universal and can be applied to analyze other similar substances by altering the matched aptamer.