组成图示
点击图片查看大图 · 依据论文自动绘制
传感器类型
微流控生物传感器
检测对象
猫杯状病毒（feline calicivirus, FCV，作为人类诺如病毒模型）；样品基质：环境水样、细胞培养裂解液（DMEM+10% FBS）
检测原理
FCV与生物素化抗FCV单抗标记的链霉亲和素脂质体结合，形成病毒–脂质体复合物。电场驱动复合物在玻璃微通道内的纳米多孔膜处预浓缩，提高局部病毒浓度并增强抗体–抗原结合效率。浓缩复合物被洗脱至下游由多克隆抗FCV抗体修饰的蛋白A超顺磁珠捕获床，未结合脂质体经洗涤去除。随后加入OG裂解捕获的脂质体，释放封装的SRB荧光染料。荧光强度随FCV浓度增加而增加，通过图像处理对裂解后下游荧光进行时间积分得到定量信号。脂质体封装大量染料并提供信号放大，预浓缩则改善结合动力学。
检测灵敏度
LOD: 1.6×10^5 PFU/mL（集成预浓缩荧光微流控器件）；LOD: 3.2×10^6 PFU/mL（无预浓缩电化学微流控器件）；LOD: 约4×10^4 PFU/mL（微孔板脂质体免疫分析）
效应效果
作者通过微孔板脂质体免疫分析筛选抗体对，最佳多克隆捕获抗体与单克隆报告抗体在5000 ng/mL FCV时信噪比接近9，并在含DMEM和10% FBS的细胞裂解液中优化封闭以降低非特异结合。集成预浓缩器件LOD为1.6×10^5 PFU/mL，比无预浓缩电化学器件的3.2×10^6 PFU/mL低一个数量级。与SERS相比，文献SERS约10^6 particles/mL（约10^4 PFU/mL）灵敏度相当或更优，但设备约15000美元且孵育约20 h；本系统检测时间约2.5 h，体积小，适合现场筛查。与微流控RT-PCR相比，可避免温度循环、酶吸附、蒸发、气泡及腐殖酸抑制。原文未报告RSD、稳定性或实际水样加标回收率。
传感器的构成
- 基底/微流控通道：玻璃微通道（glass microchannels）与原位制备纳米多孔膜（nanoporous membranes），用于电迁移预浓缩病毒–脂质体复合物
- 捕获识别层：偶联多克隆抗FCV抗体（anti-FCV pAb）的蛋白A超顺磁珠（Protein A superparamagnetic beads），在磁场下形成捕获床
- 报告识别元件：生物素化单克隆抗FCV抗体（biotinylated anti-FCV mAb），与病毒表面抗原结合并通过生物素–链霉亲和素连接脂质体
- 信号标记/放大元件：链霉亲和素偶联脂质体（streptavidin-conjugated liposomes），封装荧光染料硫磺素B（SRB）和HEPES，作为可裂解释放信号的纳米囊泡
- 可视化脂质体膜：含0.33 mol%罗丹明B磺酰基磷脂（1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)），用于观察预浓缩过程
- 裂解/信号释放剂：辛基-β-D-吡喃糖苷（OG），裂解捕获的脂质体释放SRB荧光
- 缓冲/封闭介质：HEPES-生理盐水-蔗糖缓冲液（HSS）和PBS，维持脂质体稳定并减少非特异结合；微孔板筛选中使用Tween-20封闭
- 读出系统：荧光成像与图像处理，积分裂解后下游荧光强度得到信号
中文摘要
本文报道一种集成微流控生物传感器，将样品预浓缩与脂质体信号放大相结合，用于环境水样中肠道病毒检测。该方法克服细胞培养法耗时长以及PCR法需大量处理水样以去除抑制物的问题。采用免疫夹心法，报告抗体标记于脂质体。以猫杯状病毒（FCV）作为人类诺如病毒模型，开发集成器件用于环境相关病毒检测。玻璃微通道中原位制备纳米多孔膜，并施加电场，使病毒–脂质体复合物预浓缩，从而提高抗体–病毒结合效率。浓缩复合物被洗脱至下游检测区，捕获的脂质体被裂解释放荧光染料，通过图像处理定量。与无预浓缩的优化电化学脂质体微流控生物传感器相比，集成器件对FCV的检出限为1.6×10^5 PFU/mL，比无预浓缩低一个数量级。该显著改进是将其用作环境水样病毒早期筛查系统的关键步骤。
英文摘要
An integrated microfluidic biosensor is presented that combines sample pre-concentration and liposome-based signal amplification for the detection of enteric viruses present in environmental water samples. This microfluidic approach overcomes the challenges of long assay times of cell culture-based methods and the need to extensively process water samples to eliminate inhibitors for PCR-based methods. Here, viruses are detected using an immunoassay sandwich approach with the reporting antibodies tagged to liposomes. Described is the development of the integrated device for the detection of environmentally relevant viruses using feline calicivirus (FCV) as a model organism for human norovirus. In situ fabricated nanoporous membranes in glass microchannels were used in conjunction with electric fields to achieve pre-concentration of virus-liposome complexes and therefore enhance the antibody-virus binding efficiency. The concentrated complexes were eluted to a detection region downstream where captured liposomes were lysed to release fluorescent dye molecules that were then quantified using image processing. This system was compared to an optimized electrochemical liposome-based microfluidic biosensor without pre-concentration. The limit of detection of FCV of the integrated device was at 1.6 × 10(5) PFU/mL, an order of magnitude lower than that obtained using the microfluidic biosensor without pre-concentration. This significant improvement is a key step toward the goal of using this integrated device as an early screening system for viruses in environmental water samples.