DBT-National Institute of Animal Biotechnology, Hyderabad, 500032, Telangana, India.
CSIR-Indian Institute of Chemical Biology (CSIR-IICB), Kolkata, 700091, India; IICB-Translational Research Unit of Excellence, Kolkata, 700091, India.
Biosens Bioelectron. 2019 Sep 15;141:111398. doi: 10.1016/j.bios.2019.111398. Epub 2019 May 31.
Fluorine doped tin oxide (FTO) electrochemical immunosensor has been developed for rapid detection of urokinase type plasminogen activator receptor (uPAR) - a biomarker for cancer. uPAR is a GPI-anchored cell membrane receptor that shows increased expression in many types of human cancers which include breast, prostate, colorectal, and non-small cell lung cancer. In this study, a novel ultrasensitive FTO graphene nanosheets based electrode was used as a working probe to analyze the interaction between urokinase plasminogen activator (uPA) and monoclonal uPAR antibody (Ab). Graphene nanosheets (GNS) exhibited high conductivity, thereby increasing the sensitivity of the immunochemical assay. GNS were coupled with uPAR-Ab via carbodiimide activation chemistry with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as a heterobifunctional crosslinker. The confirmation of immobilization events was done by biophysical methods such as UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), atomic force microscopy (AFM), differential pulse (DPV), and cyclic voltammetry (CV). The immobilization conditions were optimized in accordance with the best sensor response. Under optimum conditions, the proposed sensor displayed wide linear detection range (1 fM to 1 μM) with a detection limit of 4.8 fM in standard. The developed sensor was profitably engaged to detect uPA in spiked serum samples up to 9.2 pM. Furthermore, the developed uPAR immunosensor showed good reproducibility, repeatability, and storage stability (75% of initial activity observed up to 4 weeks). FTO/GNS/uPAR-Ab/uPA-Ag immunosensor displayed acceptable performance for detection of uPA and exhibited low detection limit with high reproducibility. The proposed immunosensor is 'easy to use', highly specific, and can be used as a quantitative tool making it a tenable alternate for the detection of uPAR in cancer patients.
掺氟氧化锡（FTO）电化学免疫传感器已被开发用于快速检测尿激酶型纤溶酶原激活物受体（uPAR）-一种癌症的生物标志物。uPAR 是一种 GPI 锚定的细胞膜受体，在许多类型的人类癌症中表达增加，包括乳腺癌、前列腺癌、结直肠癌和非小细胞肺癌。在这项研究中，一种新型的超灵敏 FTO 石墨烯纳米片基电极被用作工作探针，以分析尿激酶纤溶酶原激活物（uPA）与单克隆 uPAR 抗体（Ab）之间的相互作用。石墨烯纳米片（GNS）表现出高导电性，从而提高了免疫化学分析的灵敏度。GNS 通过碳二亚胺活化化学与 1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）/N-羟基琥珀酰亚胺（NHS）作为异双功能交联剂与 uPAR-Ab 偶联。通过物理方法如紫外-可见光谱、傅里叶变换红外光谱（FTIR）、扫描电子显微镜（SEM）、原子力显微镜（AFM）、差分脉冲（DPV）和循环伏安法（CV）来确认固定化事件。根据最佳传感器响应优化了固定化条件。在最佳条件下，所提出的传感器在标准条件下显示出宽的线性检测范围（1 fM 至 1 μM），检测限为 4.8 fM。该传感器被成功地用于检测加标血清样品中的 uPA，直至 9.2 pM。此外，所开发的 uPAR 免疫传感器表现出良好的重现性、重复性和储存稳定性（观察到 4 周内初始活性的 75%）。FTO/GNS/uPAR-Ab/uPA-Ag 免疫传感器对 uPA 的检测具有良好的性能，且具有低检测限和高重现性。该免疫传感器易于使用，具有高度特异性，可作为定量工具，是检测癌症患者 uPAR 的可行替代方法。
