基于双色荧光增强免疫层析技术的心肌标志物高灵敏联合即时检测方法研究

The high-sensitive simultaneous point-of-care detection of cardiac troponin I (cTnI) and creatine kinase-MB isoenzymes (CK-MB) is crucial to the accurate diagnosis of acute myocardial infarction (AMI) to reduce death rates. The bottlenecks of lateral flow immunoassay (LFIA) include its limited quantitative analysis, poor sensitivity, and the lack of multiplex detection capability. This project establishes dual-color metal-enhanced fluorescence LFIA for the high-sensitive simultaneous point-of-care detection of these two cardiac biomarkers. Ag@SiO2@QDs microbeads were prepared to provide the enhanced fluorescent signal based on the synergetic enhancement of metal-enhanced fluorescence and the large amount of adsorbed QDs to their surfaces, thus to improve the detection sensitivity. Red-color and green-color Ag@SiO2@QDs microbeads were conjugated with cTnI and CK-MB antibodies, respectively, realizing the duplex detection capacity of cardiac biomarkers on a single test line of a lateral flow strip. Moreover, a low-cost and high-performance smartphone-based dual-color LFIA readout device with an application was developed for result imaging and data analysis. Given its easy-to-use format, duplex detection capacity, and good analytical performance, the proposed dual-color metal-enhanced fluorescence LFIA platform is considered a promising point-of-care technique for AMI diagnosis.

心肌标志物肌钙蛋白I（cTnI）和肌酸激酶同工酶（CK-MB）的高灵敏联合即时检测可用于急性心肌梗塞的精准诊断，以减少急性心肌梗塞的死亡率。针对免疫层析技术缺乏定量能力、检测灵敏度差、检测通道低的瓶颈问题，本项目建立双色荧光增强免疫层析高灵敏联合即时检测心肌标志物的技术平台。合成Ag@SiO2@QDs荧光增强复合量子点微球，基于贵金属荧光增强效应和静电吸附大量量子点颗粒的“双重增强机制”显著增强荧光信号，以提高检测灵敏度。红色和绿色荧光增强复合量子点微球分别修饰cTnI和CK-MB的检测抗体，实现在单一检测线上的双通道检测能力。研制基于智能手机的低成本、高性能双色荧光免疫层析检测仪，并开发配套图像分析软件，实现检测结果的成像和数据分析。基于其简单便捷的形式，双通道检测能力和优良的分析性能，我们设计的双色荧光增强免疫层析技术平台是一种有应用前景的急性心肌梗塞即时检测技术。

免疫层析法是一种快速、低成本、用户友好的即时检测技术，并已应用于多种生化分析领域。目前，制约该技术广泛应用的瓶颈在于其缺乏定量能力、检测灵敏度差、检测通道低。采用荧光增强量子点复合微球标记的荧光免疫层析检测技术可以克服以上不足。本项目以纳米金棒AuNRs和纳米金球AuNPs作为荧光增强基底材料，制备出了AuNRs@SiO2@QDs和SiO2@AuNPs@QDs两类双色荧光增强复合量子点微球材料。基于上述材料，建立高灵敏双色荧光免疫层析检测技术，并应用于心肌标志物cTnI和CK-MB、新型冠状病毒S1和NP抗原、新型冠状病毒野生和突变位点的联合检测。抗原检测限均达到100 pg/mL以内。此外，研制小型化、低成本的手持式和无线式双色荧光免疫层析检测仪，并开发配套软件。本研究建立了一种快速、高灵敏、多通道的免疫层析检测平台，有望在资源有限的条件下实现对于心肌标志物、病原体等的即时检测。