Amity Institute of Biotechnology, Amity University Maharashtra - Mumbai - Pune Expressway, Bhatan, Panvel, Maharashtra 410206, India.
Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India; Homi Bhabha National Institute, Anushaktinagar, Mumbai 400085, India.
Int J Biol Macromol. 2023 Sep 30;249:125934. doi: 10.1016/j.ijbiomac.2023.125934. Epub 2023 Jul 22.
Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP, as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP. Thus, heparinase leads to dissociation of TMPyP aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP-heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited.
肝素酶是唯一一种能将常用的抗凝血剂肝素分解成具有治疗意义的低分子量肝素的哺乳动物内切糖苷酶。重要的是，肝素酶已被认为是一种恶性疾病的诊断标志物。因此，开发肝素酶的检测方案至关重要。然而，光学方法在肝素酶测定方面受到限制。在本工作中，我们报道了一种基于肝素模板聚合的四阳离子卟啉衍生物 TMPyP 的荧光传感器，用于检测肝素酶。肝素酶可裂解肝素结构中氨基葡萄糖和糖醛酸之间的糖苷键，破坏其原有的聚电解质性质，导致 TMPyP 聚集。因此，肝素酶会导致 TMPyP 聚集物的解离，并产生光学信号。该系统对肝素酶具有灵敏和选择性的响应，检测限（LOD）为 0.3 pmol/L。此外，通过利用 OSCS 是肝素酶活性的抑制剂这一事实，该系统还可用于检测痕量的肝素中的过硫酸化软骨素硫酸盐（OSCS），OSCS 是肝素的一种掺杂物，这会导致 TMPyP-肝素-肝素酶体系的单体/聚集平衡的光物理特征发生反向调制。通过基态吸收、稳态发射和时间分辨发射测量对传感机制进行了彻底的证明。在肝素酶选择性研究中，使用溶菌酶、α-淀粉酶、胃蛋白酶、胰蛋白酶、脂肪酶和葡萄糖氧化酶对传感器的选择性进行了测试，并使用文献中报道的另一种方法对该方法进行了验证。该研究为开发光学方法检测肝素酶和过硫酸化软骨素提供了一种新的途径，目前这方面的方法受到限制。
