Xu Jianglian, Zhang Han, Tan Yongfeng, Gao Zhuangqiang, Ye Haihang, Dong Haifeng
Marshall Laboratory of Biomedical Engineering, Shenzhen Key Laboratory for Nano-Biosensing Technology, Department of Biomedical Engineering, Medical School, Shenzhen University, Shenzhen, Guangdong, 518060, PR China.
Marshall Laboratory of Biomedical Engineering, Shenzhen Key Laboratory for Nano-Biosensing Technology, Department of Biomedical Engineering, Medical School, Shenzhen University, Shenzhen, Guangdong, 518060, PR China.
Talanta. 2025 May 1;286:127432. doi: 10.1016/j.talanta.2024.127432. Epub 2024 Dec 25.
Colorimetric enzyme-linked immunosorbent assays (CELISAs) have long been used for protein biomarker detection in diagnostics. Unfortunately, as confined by the monochromatic nature of detection signals and the limited catalytic activity of enzymes, CELISAs suffer from poor visual resolution and low sensitivity, hindering their effectiveness for early diagnostics in resource-limited settings. Herein, we report an ultrasensitive, high-visual-resolution CELISA (named PE-TSA-AuAg Cage-CELISA) that combines kinetically controlled growth of Ag in AuAg nanocages with poly-enzyme-boosted tyramide signal amplification (PE-TSA), enabling visual semiquantitative detection of protein biomarkers at attomolar levels with the naked eye. Specifically, the assay begins with the formation of sandwich-type immunocomplexes on a microplate in the presence of targets, and the labeled poly-horseradish peroxidases (poly-HRPs) initiate TSA, resulting in attaching numerous alkaline phosphatases (ALPs) on the microplate. The ALPs further catalyze ascorbic acid 2-phosphate to produce ascorbic acid, triggering the kinetically controlled growth of Ag inside AuAg nanocages. This process induces vivid multicolor variations spanning the visible spectrum range of 691∼477 nm, allowing for visual semiquantitation of protein biomarkers at ultralow levels without requiring specialized equipment. Using interleukin-12 as a model protein biomarker, we demonstrate that the PE-TSA-AuAg Cage-CELISA achieves a visual semiquantitative limit of detection (LOD) of 5 fg mL (67 aM) and an instrumental quantitative LOD of 0.71 fg mL (9.5 aM), representing an 853-fold improvement compared to the conventional HRP-based CELISA. Our findings suggest that the PE-TSA-AuAg Cage-CELISA has the potential to serve as an affordable and effective biosensing platform for early diagnostics in resource-limited settings.
比色酶联免疫吸附测定法（CELISAs）长期以来一直用于诊断中的蛋白质生物标志物检测。不幸的是，由于检测信号的单色性质以及酶的催化活性有限，CELISAs的视觉分辨率差且灵敏度低，这阻碍了它们在资源有限环境中进行早期诊断的有效性。在此，我们报告了一种超灵敏、高视觉分辨率的CELISA（名为PE-TSA-AuAg笼状CELISA），它将AuAg纳米笼中Ag的动力学控制生长与多酶增强的酪胺信号放大（PE-TSA）相结合，能够用肉眼对阿托摩尔水平的蛋白质生物标志物进行视觉半定量检测。具体而言，该测定法首先在存在靶标的情况下在微孔板上形成夹心型免疫复合物，标记的多辣根过氧化物酶（poly-HRPs）启动TSA，导致在微孔板上附着大量碱性磷酸酶（ALPs）。ALPs进一步催化抗坏血酸2-磷酸产生抗坏血酸，触发AuAg纳米笼内Ag的动力学控制生长。这个过程会引起跨越691∼477 nm可见光谱范围的鲜明多色变化，无需专门设备即可对超低水平的蛋白质生物标志物进行视觉半定量。以白细胞介素-12作为模型蛋白质生物标志物，我们证明PE-TSA-AuAg笼状CELISA实现了5 fg mL（67 aM）的视觉半定量检测限（LOD）和0.71 fg mL（9.5 aM）的仪器定量LOD，与传统的基于HRP的CELISA相比提高了853倍。我们的研究结果表明，PE-TSA-AuAg笼状CELISA有潜力作为一种经济有效的生物传感平台，用于资源有限环境中的早期诊断。
