Ströhle Gisela, Goodrum Rebecca, Li Huiyan
School of Engineering, University of Guelph, Guelph, ON N1G 2W1, Canada.
Biosensors (Basel). 2025 May 6;15(5):294. doi: 10.3390/bios15050294.
Extracellular vesicles (EVs) have emerged as promising biomarkers and therapeutic agents, yet their quantification remains technically challenging due to the limitations of conventional methods. Here, a low-cost, fluorescence-based, paper-strip immunoassay is presented for rapid and semi-quantitative estimation of EV concentration, inspired by pH strips. The assay utilizes nitrocellulose membranes functionalized with capture antibodies (anti-CD63, CD9, CD81) and fluorescent dye (ExoBrite™) for EV detection. Systematic optimization of assay parameters-including dye application sequence, incubation time, antibody configuration, and dye concentration-revealed that labeling EVs with dye and incubating on the nitrocellulose paper strips for 20 min yielded the strongest and most reproducible signal. A 200× dilution of ExoBrite™ dye was determined to provide the best balance between sensitivity and specificity. A standard curve generated through twofold serial dilution of EVs from ovarian cancer cell culture medium confirmed a positive, concentration-dependent fluorescence response, establishing a usable dynamic range. Compared to existing technologies, this platform enables fast, simple-to-implement EV quantification using minimal sample volume and equipment. The simplicity and scalability of the method offer strong potential for use in clinical diagnostics and EV research applications.
细胞外囊泡（EVs）已成为有前景的生物标志物和治疗剂，但由于传统方法的局限性，其定量在技术上仍然具有挑战性。在此，受pH试纸启发，提出了一种基于荧光的低成本纸条免疫分析法，用于快速半定量估计EV浓度。该分析法利用用捕获抗体（抗CD63、CD9、CD81）和荧光染料（ExoBrite™）功能化的硝酸纤维素膜进行EV检测。对分析参数（包括染料应用顺序、孵育时间、抗体配置和染料浓度）的系统优化表明，用染料标记EV并在硝酸纤维素纸条上孵育20分钟可产生最强且最可重复的信号。确定ExoBrite™染料200倍稀释可在灵敏度和特异性之间提供最佳平衡。通过对卵巢癌细胞培养基中的EV进行两倍系列稀释生成的标准曲线证实了阳性的浓度依赖性荧光响应，建立了可用的动态范围。与现有技术相比，该平台使用最少的样品量和设备就能实现快速、易于实施的EV定量。该方法的简单性和可扩展性在临床诊断和EV研究应用中具有很大的应用潜力。
