组成图示
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传感器类型
压电(QCM)生物传感器
检测对象
抗瓜氨酸化肽抗体（anti-citrullinated peptide antibodies）、类风湿关节炎特异性自身抗体（RA-specific autoantibodies）；样品基质：人血清
检测原理
该传感器以QCM晶片为压电换能器，SWNT薄膜提供三维抗原负载平台，Tween-COOH作为间隔和连接层，将瓜氨酸化环状角蛋白肽固定于表面。人血清稀释后注入，血清中抗瓜氨酸化肽抗体与固定肽特异性结合。抗体结合使传感器表面质量增加，导致石英晶体共振频率下降；冲洗后记录净频率变化作为信号。抗体浓度越高，结合量越大，频率下降越明显。方法为无标记检测，不需要二次抗体或荧光标记，并可通过监测耗散判断结合状态。
检测灵敏度
Detection levels were as low as 89 fmol of specific antibody.
效应效果
该传感器可直接检测未预处理人血清，抗瓜氨酸化肽与非瓜氨酸化肽反应差异显著（p<0.0001），正常与骨关节炎对照无显著差异。批间变异<14%，批内变异<15%。单次运行约可测22份RA和7份对照血清，抗原负载约3 ng。与ELISA和微阵列相比，纳米管法检出23/32（71.8%）RA阳性，高于ELISA 12/32（37.5%）和微阵列11/32（34.3%）；灵敏度71.9%，特异性95.8%，PPV 95.8%，NPV 71.9%，准确性0.94，均优于ELISA（0.64）和微阵列（0.54）。作者认为可用于诊断、研究、个体化治疗和药物筛选。
传感器的构成
- 基底/换能器：5 MHz AT-cut 金膜石英晶体微天平（QCM）传感晶片，提供压电换能与频率读出
- 纳米材料修饰层：单壁碳纳米管（SWNT）薄膜，氯仿滴涂并60°C烘烤，提供三维高表面负载平台
- 间隔/连接层：羧基化Tween20（Tween-COOH，含PEG单元），降低非特异结合并隔离疏水纳米管对肽的变性
- 化学偶联层：EDC/NHS活化Tween-COOH羧基，用于共价固定抗原
- 识别元件：瓜氨酸化环状角蛋白肽（citrullinated cyclic filaggrin peptide）作为RA特异性抗原，非瓜氨酸化肽作对照
- 封闭层：3%胎牛血清（FCS）/0.1% Tween20磷酸盐缓冲液，封闭未结合位点降低背景
- 样品/信号事件：人血清稀释1/300注入，抗瓜氨酸化肽抗体结合抗原，质量增加引起QCM频率下降
中文摘要
本文报道了一种无标记肽包覆碳纳米管免疫传感器，用于直接检测人血清。将类风湿关节炎（RA）特异性瓜氨酸化环状肽固定在功能化单壁碳纳米管上，再沉积于石英晶体微天平（QCM）传感晶片。用RA患者血清探测该纳米管传感器，通过QCM检测抗体结合。通过与正常血清、疾病对照血清以及未修饰天然肽的比较，确认了特异性抗体结合。该纳米管传感器具有飞摩尔级检测灵敏度，高于已建立的ELISA和近期报道的微阵列检测系统，分别比ELISA和微阵列多检出34.4%和37.5%的抗瓜氨酸化肽抗体阳性RA患者。与ELISA或微阵列相比，阴性结果真实提示未患RA的概率分别提高18.4%和19.6%。该无标记生物传感器的性能使其可用于血清直接检测的研究与诊断。
英文摘要
We demonstrate a label-free peptide-coated carbon nanotube-based immunosensor for the direct assay of human serum. A rheumatoid arthritis (RA)-specific (cyclic citrulline-containing) peptide, was immobilized to functionalized single-walled carbon nanotubes deposited on a quartz crystal microbalance (QCM) sensing crystal. Serum from RA patients was used to probe these nanotube-based sensors, and antibody binding was detected by QCM sensing. Specific antibody binding was also determined by comparing the assay of two serum control groups (normal and diseased sera), and the native unmodified peptide. The sensitivity of the nanotube-based sensor (detection in the femtomol range) was higher than that of the established ELISA and recently described microarray assay systems, detecting 34.4 and 37.5% more RA patients with anti-citrullinated peptide antibodies than those found by ELISA and microarray, respectively. There was also an 18.4 and 19.6% greater chance of a negative test being a true indicator of a person not having RA than by either ELISA or microarray, respectively. The performance of our label-free biosensor enables its application in the direct assay of sera in research and diagnostics.