基于荧光肽探针的肝素及其过度硫酸化软骨素硫酸酯污染物的高灵敏比率检测
Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.
机构信息
Center for Design and Applications of Molecular Catalysts, Department of Chemistry and Chemical Engineering, Inha University, Incheon 402-751, South Korea.
Center for Design and Applications of Molecular Catalysts, Department of Chemistry and Chemical Engineering, Inha University, Incheon 402-751, South Korea.
出版信息
Biosens Bioelectron. 2017 May 15;91:545-552. doi: 10.1016/j.bios.2017.01.002. Epub 2017 Jan 4.
The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R=0.99) in aqueous solutions, 2.98nM (R=0.98) in 1% serum samples, and 3.43nM (R=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.
肝素是临床上常用的抗凝剂，同时也是其污染物过硫酸化硫酸软骨素（OSCS），对其进行选择性和灵敏的检测非常重要。我们首次报道了一种比率传感方法，用于使用荧光肽探针（Pep1，芘-GSRKR）和肝素消化酶检测肝素以及肝素中的 OSCS 污染物。Pep1 在水溶液中对肝素的纳摩尔浓度表现出高度灵敏的比率响应，在较宽的 pH 范围（2~11）内，并且对肝素具有高度选择性的比率响应，对透明质酸和硫酸软骨素等生物竞争物具有很高的选择性。Pep1 在水溶液中和人血清样品中对纳摩尔浓度的肝素表现出线性的比率响应。在水溶液中，肝素的检测限计算为 2.46nM（R=0.99），在 1%血清样品中为 2.98nM（R=0.98），在 5%血清样品中为 3.43nM（R=0.99）。Pep1 分别与肝素酶 I、II 和 III 一起用于检测肝素中的污染 OSCS。使用 Pep1 和肝素酶 II 的比率传感方法对于检测肝素中的 OSCS 污染物非常灵敏、快速且高效。Pep1 与肝素酶 II 结合，通过比率响应可以检测到肝素中低至 0.0001%（w/w）的 OSCS。
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