Chen Daqi, Zhu Zhuobin, Guo Wenjie, Wang Yupeng, Yu Zhiyong, Zhu Baian, Lu Jiandong, Zan Jie
School of Mechanical and Electrical Engineering, Guangzhou University, Guangzhou, Guangdong, China.
School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, Guangdong, China.
Biosens Bioelectron. 2025 Jun 15;278:117318. doi: 10.1016/j.bios.2025.117318. Epub 2025 Feb 26.
Nanopore technology is a promising single-molecule sensing platform that can identify substances through the precise monitoring of changes in ion currents. However, protein detection in clinical samples using solid-state nanopores remains challenging due to their heterogeneously charged spherical structure, which results in signals with extremely low signal-to-noise ratios (SNR) and low capture rates that are difficult to analyze. In this study, we employed a double-antibody sandwich technique to specifically capture and amplify the target antigen, which significantly improves the SNR and effectively distinguishes the target signal from background interference. Key factors including buffer composition, voltage, antibody concentration, and pore dimensions were systematically optimized to further improve capture efficiency. The optimized approach enabled precise and reliable detection of retinol-binding protein 4 (RBP4) with an excellent linear response within the range of 55 fM to 5.5 pM. Moreover, our method facilitates quantitative detection of RBP4 in clinical urine samples within 40 min, and achieves 100% accuracy in distinguishing between 11 urine samples from chronic kidney disease (CKD) patients and healthy donors, highlighting its robustness and specificity. Our research not only paves a new pathway for efficient RBP4 detection, but also provides valuable insights into the application of nanopore technology for the clinical diagnosis of protein biomarkers.
纳米孔技术是一个很有前景的单分子传感平台，它可以通过精确监测离子电流的变化来识别物质。然而，由于临床样本中的蛋白质具有异质带电的球形结构，使用固态纳米孔进行蛋白质检测仍然具有挑战性，这会导致信号的信噪比极低，捕获率也很低，难以分析。在本研究中，我们采用双抗体夹心技术特异性捕获并放大目标抗原，这显著提高了信噪比，并有效地区分目标信号与背景干扰。我们系统地优化了包括缓冲液组成、电压、抗体浓度和孔尺寸在内的关键因素，以进一步提高捕获效率。优化后的方法能够精确可靠地检测视黄醇结合蛋白4（RBP4），在55飞摩尔至5.5皮摩尔范围内具有出色的线性响应。此外，我们的方法能够在40分钟内对临床尿液样本中的RBP4进行定量检测，在区分11份慢性肾病（CKD）患者和健康供体的尿液样本时准确率达到100%，突出了其稳健性和特异性。我们的研究不仅为高效检测RBP4开辟了一条新途径，还为纳米孔技术在蛋白质生物标志物临床诊断中的应用提供了有价值的见解。
