Karbalaee Zahra, Rezayan Ali Hossein, Taheri Ramezan Ali, Mirhosseini Seyed Ali, Barshan-Tashnizi Mohammad
Department of Nanobiotechnology and Biomimetics, School of Bioengineering, College of Interdisciplinary Science and Technology, University of Tehran, Tehran, Iran.
Nanobiotechnology Research Center, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Sci Rep. 2025 May 16;15(1):17024. doi: 10.1038/s41598-025-01686-9.
This study demonstrates enhanced detection of Shiga toxin (Stx), a key virulence factor in Shigella dysenteriae-induced bloody diarrhea, through optimized surface plasmon resonance (SPR) biosensor design. We present a comparative evaluation of antibody immobilization strategies, revealing significant advantages of protein G-mediated oriented immobilization over conventional covalent attachment. The covalent (non-oriented) approach using 11-mercaptoundecanoic acid-modified chip showed moderate performance (K = 37 nM, LOD = 28 ng/mL). In contrast, protein G-assisted orientation dramatically improved detection capabilities, achieving a 2.9-fold lower detection limit (9.8 ng/mL) and 2.3-fold higher binding affinity (K = 16 nM). Control measurements with free antibody-antigen interactions established a baseline affinity (K = 10 nM), demonstrating that the oriented method preserves 63% of native binding efficiency versus only 27% in the covalent approach. Mechanistic studies attribute these improvements to protein G's ability to maintain optimal antibody orientation, thereby: (1) maximizing paratope accessibility, (2) minimizing steric interference, and (3) preserving binding site functionality. The 57% reduction in K relative to covalent immobilization confirms the method's efficacy in maintaining antibody performance post-immobilization. These findings establish protein G-mediated orientation as the superior strategy for SPR-based Stx detection, offering substantial improvements in sensitivity and reliability for clinical diagnostics and food safety applications. The approach demonstrates particular promise for rapid, label-free detection of bacterial toxins in resource-limited settings.
本研究表明，通过优化表面等离子体共振（SPR）生物传感器设计，可增强对志贺毒素（Stx）的检测，志贺毒素是痢疾志贺氏菌引起血性腹泻的关键毒力因子。我们对抗体固定策略进行了比较评估，结果显示，与传统的共价连接相比，蛋白G介导的定向固定具有显著优势。使用11-巯基十一烷酸修饰芯片的共价（非定向）方法表现中等（K = 37 nM，检测限 = 28 ng/mL）。相比之下，蛋白G辅助定向显著提高了检测能力，检测限降低了2.9倍（9.8 ng/mL），结合亲和力提高了2.3倍（K = 16 nM）。通过游离抗体-抗原相互作用的对照测量确定了基线亲和力（K = 10 nM），这表明定向方法保留了63%的天然结合效率，而共价方法仅保留了27%。机理研究将这些改进归因于蛋白G保持最佳抗体定向的能力，从而：（1）最大限度地提高互补位可及性，（2）最小化空间位阻干扰，（3）保留结合位点功能。相对于共价固定，K值降低了57%，证实了该方法在固定后维持抗体性能方面的有效性。这些发现确立了蛋白G介导的定向作为基于SPR的Stx检测的优越策略，为临床诊断和食品安全应用的灵敏度和可靠性提供了实质性改进。该方法对于在资源有限的环境中快速、无标记检测细菌毒素具有特别的前景。
