组成图示
示意图生成中
传感器类型
电化学生物传感器
检测对象
丙型肝炎病毒RNA（HCV RNA，基因型1、2A/C、2B、3）；样品基质：血清
检测原理
该传感器基于DNA杂交与酶促电化学信号放大。血清HCV RNA先经RT-PCR扩增，并用生物素标记引物生成生物素化cDNA。cDNA与固定在石墨电极PPO/STA薄膜上的HCV特异性生物素化寡核苷酸探针杂交；随后亲和素-辣根过氧化物酶（AV-HRP）结合cDNA上的生物素，使HRP靠近电极。在0.1 mol L-1 PBS（pH 7.0）中加入H2O2和KI，HRP催化H2O2氧化I-生成I2。I2在石墨电极上于-0.45 V（vs Ag/AgCl）被还原，产生阴极电流。HCV扩增产物越多，结合AV-HRP越多，I2生成量越大，安培电流越强；阴性样品仅产生接近背景的电流。RT-PCR提供核酸扩增，酶催化提供信号放大。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率及相关系数。
效应效果
BSA封闭降低PPO薄膜对生物素-HRP的非特异吸附。优化条件为STA 20 μg mL-1、HCV 1探针40倍稀释。阴性均值-0.37 ± 0.05 μA，阈值-0.47 μA；阳性样品至少为背景4倍。8例血清与Amplicor HCV试剂盒比较：HCV 1探针下阳性样本1–5接近阳性对照，阴性样本6–8接近阴性对照；HCV 2A/C、2B、3探针下除样本7外均低于阈值，样本7在HCV 3探针下电流0.863 ± 0.006 μA，约为阈值2倍。阳性与阴性对照重复5次，RSD分别为0.2%和1.2%。作者认为该传感器可用于HCV RNA检测与分型。
传感器的构成
- 基底/换能器电极：石墨电极（graphite electrode），作为工作电极与电化学换能器。
- 溶胶-凝胶修饰层：硅氧烷-聚环氧丙烷（siloxane-poly(propylene oxide), PPO）杂化薄膜，由3-(异氰酸丙基)三乙氧基硅烷（IsoTrEOS）与O,O'-双(2-氨基丙基)聚环氧丙烷（PPO，MW 4000）经溶胶-凝胶法制备并浸涂沉积，厚度约5.0 μm，用于包埋链霉亲和素。
- 识别元件：链霉亲和素（streptavidin, STA）包埋于PPO薄膜中，通过生物素-亲和素作用固定生物素化寡核苷酸探针。
- 识别探针：5'-生物素化18-mer HCV特异性寡核苷酸探针（biotinylated 18-mer oligonucleotide probes，HCV 1、2A/C、2B、3），与目标cDNA杂交。
- 封闭剂：0.1%牛血清白蛋白（BSA）在0.1 mol L-1 PBS pH 7.0中封闭，减少非特异吸附。
- 信号标记物：亲和素-辣根过氧化物酶（avidin-peroxidase, AV-HRP）偶联物，结合生物素标记扩增产物。
- 底物/电子供体：过氧化氢（H2O2）和碘化钾（KI）在0.1 mol L-1 PBS pH 7.0中，HRP催化I-氧化为I2。
中文摘要
本文报道了一种新型恒电位安培DNA生物传感器，用于丙型肝炎病毒（HCV）RNA检测与基因分型（1、2A/C、2B、3）。作者用溶胶-凝胶法制备硅氧烷-聚环氧丙烷（PPO）杂化薄膜，将链霉亲和素（STA）包埋其中并浸涂于石墨电极表面；生物素化18-mer寡核苷酸探针经STA固定。血清HCV RNA经RT-PCR扩增得到生物素标记cDNA，与电极上HCV特异性探针杂交后，加入亲和素-辣根过氧化物酶（AV-HRP）偶联物。在0.1 mol L-1 PBS（pH 7.0）中，以H2O2和KI为底物，在-0.45 V（vs Ag/AgCl）进行恒电位安培检测。HCV RNA阴/阳性对照及患者血清均被分析，并与商品化试剂盒比较。结果表明，该方法适合STA固定和HCV诊断。
英文摘要
Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45V versus Ag/AgCl using H(2)O(2) and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease.