组成图示
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传感器类型
电化学生物传感器
检测对象
人白细胞介素-2互补寡核苷酸（chIL-2, complementary human IL-2 oligonucleotide）；样品基质：缓冲液中的DNA溶液（0.5 M醋酸缓冲液pH 4.80杂交，0.1 M PBS pH 7.00检测）
检测原理
传感器以CPE为基底，经电化学活化后在负电位下吸附hIL-2单链探针。目标chIL-2与探针杂交后形成dsDNA。BCB为阳离子平面染料，可经静电作用结合DNA磷酸骨架并嵌入碱基间；ssDNA结构松散，BCB结合量高，DPV还原峰强；杂交形成dsDNA后，BCB可及性降低或空间位阻增加，结合量减少，还原峰下降。因此BCB峰电流变化反映杂交程度。对探针固定量而言，BCB峰电流随hIL-2浓度增加而增大，在0.01–0.20 μM呈线性；摘要亦报告目标寡核苷酸在1.0×10−8–5.0×10−6 M线性。检测通过DPV读取BCB还原峰，未采用HCR/RCA等放大策略。
检测灵敏度
LOD: 9.00 nM；线性范围: 1.0×10−8 to 5.0×10−6 M（摘要，目标寡核苷酸）；线性范围: 0.01–0.20 μM（探针固定，Fig. 7B）；斜率: 119.45；R^2 = 0.9982
效应效果
传感器对互补目标chIL-2选择性良好：探针修饰电极BCB还原峰为33 μA，杂交后降至9.2 μA；非互补序列16SR、YF270和HgbBF未引起显著信号下降。t检验给出tcalculated分别为6.36、2.84和15.29，与tcritical 2.13（p=0.05）和3.75（p=0.01）比较，作者认为互补与非互补信号差异显著。每次测量使用新鲜CPE表面，通过切割抛光更新电极，未报告RSD、实际样品回收率或与ELISA/HPLC/qPCR的对比。作者认为CPE低成本、易制备、可再生，BCB可作为电活性指示剂用于短DNA序列杂交检测。
传感器的构成
- 基底/换能器电极：碳糊电极（CPE，石墨粉与石蜡油70:30 w/w），工作电极，表面可更新
- 电极活化层：0.50 M醋酸缓冲液（pH 4.80）含20 mM NaCl，+0.20 V vs SCE 5 min电化学活化，提高探针吸附与BCB富集
- 识别元件：hIL-2反义寡核苷酸探针（20-mer，1 μM），-0.50 V vs SCE 5 min吸附固定
- 杂交层：chIL-2互补寡核苷酸（1 μM），+0.50 V vs SCE 5 min杂交形成dsDNA
- 信号标记物：亮甲酚蓝（BCB，1 mM）阳离子电活性染料，与ssDNA/dsDNA静电结合并产生DPV还原峰
- 检测介质：0.1 M磷酸缓冲液（pH 7.00）用于DPV测量；0.5 M醋酸缓冲液（pH 4.80）含20 mM NaCl用于固定与杂交
- 参比/辅助电极：饱和甘汞电极（SCE）和铂丝辅助电极，组成三电极体系
中文摘要
本文报道了一种基于碳糊电极（CPE）的电化学DNA生物传感器，用于固定和检测短DNA序列，并以亮甲酚蓝（BCB）作为电活性标记。BCB与DNA的结合通过电化学方法检测，BCB与单链DNA（ssDNA）和双链DNA（dsDNA）相互作用时呈现不同信号，其信号变化可反映电极表面杂交程度。研究考察了溶液pH对BCB电化学行为、BCB在CPE上富集以及差分脉冲伏安法（DPV）信号的影响，pH 7.00时BCB信号最高。同时优化了CPE电化学预处理、探针固定电位和时间、BCB富集条件。BCB峰电流与目标寡核苷酸浓度在1.0×10−8至5.0×10−6 M范围内呈线性关系，检出限为9.00 nM。利用非互补寡核苷酸评价了传感器选择性。
英文摘要
A new electrochemical DNA biosensor is presented based on carbon past electrode (CPE) for immobilization and detection of short DNA sequences with brilliant cresyl blue (BCB) as electroactive label. The interaction of BCB with DNA is electrochemically detected and BCB displays different signals in the interaction to ssDNA and dsDNA and variation in the BCB signal represents the extent of hybridization at the electrode surface. The effect of solution pH on electrochemical behavior of BCB was investigated. Additionally, the effect of solution pH on BCB accumulation on the CPE was studied. Furthermore, experiments showed that the solution pH could influence the differential pulse voltammetry (DPV) signal of BCB accumulated on the electrode and the highest BCB signal was obtained in pH 7.00. The effect of electrochemical pretreatment of CPE on the ability of electrode in probe adsorption, BCB accumulation and conditions of probe immobilization including potential and time was investigated and optimum conditions were suggested. The peak currents of BCB were linearly related to the concentration of the target oligonucleotide sequence in the range of 1.0x10(-8) to 5.0x10(-6)M. The detection limit of this approach was 9.00nM. The selectivity of the biosensor was studied using noncomplementary oligonucleotide.