State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
OPLI (Suzhou) Biotechnology Company Limited, New District, Suzhou 215163, China.
Anal Chem. 2024 Aug 20;96(33):13482-13493. doi: 10.1021/acs.analchem.4c01646. Epub 2024 Aug 2.
Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.
纤溶活性测定在心血管疾病的检测、诊断和治疗以及纤溶药物的开发中尤为重要。本研究提出了一种新颖的基于有序多孔层干涉仪（OPLI）的实时、无标记动态检测纤溶活性的有效策略。将纤维蛋白或纤维蛋白与纤溶酶原（Plg）的混合物载入高度有序的硅溶胶胶体晶体（SCC）薄膜支架中，构建纤溶反应干扰层，以不同的作用机制测量纤溶活性。纤溶酶触发的纤维蛋白溶解导致干涉图中的干涉条纹迁移，可通过 OPLI 系统实时跟踪光学厚度变化（ΔOT）来表示。对纤溶反应干扰层的形貌和光学特性进行了表征，并对纤溶反应干扰层中的 Plg 含量和系统的实验参数进行了优化。该方法对蚓激酶和链激酶的纤溶活性具有足够的灵敏度，线性范围分别为 12-6000 和 10-2000 U/mL。与传统的纤维蛋白平板法相比，该方法具有更低的检测限和更高的线性度。通过实时记录这两种纤溶药物模型的纤溶动力学全过程，计算出米氏常数和表观动力学参数。重要的是，该系统对其他一些血液蛋白的干扰较小，在真实全血样本的纤维蛋白活性检测中具有可靠性。本研究建立了一种更好、更有针对性的体外纤溶研究方法，为全血纤溶活性分析提供了动态监测数据。
