Oh Doogie, Chae Yun Jin, Teoh Jie Ying, Yim Bora, Yoo Dongwon, Park Yongdoo, Kim Jongseong
R&D Center, Scholar Foxtrot Co. Ltd., Seoul 02796, Republic of Korea.
Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea.
ACS Omega. 2024 Mar 5;9(11):13418-13426. doi: 10.1021/acsomega.4c00010. eCollection 2024 Mar 19.
The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein-protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings.
促血栓形成标志物的检测对于理解血栓栓塞和评估抗凝药物的疗效至关重要。α-凝血酶是一种在凝血级联过程中起关键作用的标志物。然而，在临床环境中，由于缺乏有效的检测方法，这种酶分子的检测受到了阻碍。此前，我们报道过一种α-凝血酶与糖蛋白Ib（GPIbα）的两条α链相互作用，即多价蛋白结合（MPB），采用了生物响应水凝胶纳米颗粒（纳米凝胶）和光学显微镜技术。在本研究中，我们证明了基于GPIbα的平台能够在各种诊断系统中实现对α-凝血酶的高灵敏度和定量检测。首先，开发了一种基于生物响应纳米凝胶的表面等离子体共振（nSPR）检测方法，该方法可对α-凝血酶与GPIbα的MPB作出响应。使用酶联免疫吸附测定法（一种蛋白质检测的金标准技术）进一步验证了利用GPIbα检测α-凝血酶的方法。此外，还开发了GPIbα功能化的乳胶珠来进行乳胶凝集（LA）检测，这种检测方法在医院诊断仪器中广泛使用。值得注意的是，与我们之前的光学显微镜方法相比，nSPR和LA检测对α-凝血酶检测的灵敏度提高了近1000倍。我们基于GPIbα的平台的优势在于通过蛋白质-蛋白质相互作用对α-凝血酶检测具有稳定性。相比之下，依赖于使用底物的α-凝血酶酶活性的检测方法面临着样品采集后迅速下降的挑战。这些结果表明，α-凝血酶与GPIbα的MPB是临床α-凝血酶检测的理想模式，特别是在门诊环境中。
