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
肠耶尔森菌（Yersinia enterocolitica）特定基因序列ssDNA、肠耶尔森菌LAMP扩增产物；样品基质：猪肉肉样（pork meat）DNA提取液/LAMP产物溶液
检测原理
该传感器以CILE为基底，涂覆CTS/nano-V2O5/MWCNTs复合膜。CTS带正电荷，通过静电作用吸附ssDNA探针；nano-V2O5和MWCNTs增大电极有效面积并加速电子转移。目标ssDNA与探针杂交后形成dsDNA，MB与dsDNA结合并在电极表面积累。DPV测量时，MB发生还原反应产生阴极峰电流；目标序列浓度越高，形成的dsDNA越多，结合的MB越多，MB还原峰电流差越大。实际样品中，先以LAMP在65 ℃等温扩增肠耶尔森菌gyrB基因，产物变性后与探针杂交，实现核酸信号放大。
检测灵敏度
LOD: 1.76 × 10−12 mol L−1 (3σ)；线性范围: 1.0 × 10−11–1.0 × 10−6 mol L−1；灵敏度斜率: 77.513 ± 2.325（I/μA = 77.513 ± 2.325 log[C/(mol L−1)] + 5.946 ± 0.178）；相关系数: r = 0.994；R.S.D. = 3.0%
效应效果
该传感器对互补ssDNA响应最高，对单碱基错配、三碱基错配和非互补序列响应明显降低，并能通过MB峰电流变化区分单碱基与三碱基错配序列。峰电流相对标准偏差为3.0%；4 ℃保存10天后仍保持97.4%初始灵敏度。猪肉肉样中肠耶尔森菌LAMP产物检测结果满意。与ZrO2/纳米金、ZrO2/Au和MWCNTs/纳米ZrO2修饰GCE相比，本工作检出限更低（1.76×10−12 mol/L对3.1×10−11、1.0×10−10、7.5×10−11 mol/L）且线性范围更宽。作者认为其制备简单、成本低、响应快、选择性好，可用于特定基因序列检测。
传感器的构成
- 基底电极：碳离子液体电极（CILE），由石墨粉（graphite powder）和N-己基吡啶六氟磷酸酯（HPPF6）离子液体粘结剂构成，提供高导电性和电化学活性
- 纳米复合修饰层：壳聚糖（CTS）/五氧化二钒纳米带（nano-V2O5）/多壁碳纳米管（MWCNTs）复合膜，涂覆于CILE表面；CTS成膜并静电吸附DNA，nano-V2O5和MWCNTs增大表面积并加速电子转移
- 识别元件：单链DNA探针（probe ssDNA），吸附于CTS–V2O5–MWCNTs/CILE表面，特异性识别目标ssDNA
- 杂交产物：双链DNA（dsDNA），探针与目标ssDNA杂交形成，作为MB结合位点
- 信号标记物：亚甲基蓝（MB），与dsDNA结合并在DPV中产生可还原电化学信号
- 检测缓冲液：50 mmol/L Tris–HCl缓冲液（pH 7.4），用于探针固定、杂交和DPV测量
中文摘要
本文以五氧化二钒纳米带（nano-V2O5）、多壁碳纳米管（MWCNTs）和壳聚糖（CTS）复合膜修饰碳离子液体电极（CILE），构建电化学DNA生物传感器。CILE由石墨粉和N-己基吡啶六氟磷酸酯（HPPF6）制备。复合膜用于固定单链DNA（ssDNA）探针，与目标ssDNA杂交后，用亚甲基蓝（MB）监测反应。nano-V2O5与MWCNTs协同提高ssDNA负载量和电化学响应。该传感器结合V2O5生物相容性、MWCNTs电子转移、CTS成膜和CILE高导电优势。最优条件下，差分脉冲伏安法（DPV）在1.0×10−11–1.0×10−6 mol/L范围内检测ssDNA，检出限1.76×10−12 mol/L（3σ）。传感器稳定性好，可区分单碱基和三碱基错配序列，并检测猪肉中肠耶尔森菌LAMP产物，具有特定基因序列检测潜力。
英文摘要
A new electrochemical DNA biosensor was fabricated by using a V(2)O(5) nanobelts (nano-V(2)O(5)), multi-walled carbon nanotubes (MWCNTs) and chitosan (CTS) nanocomposite materials modified carbon ionic liquid electrode (CILE) as the working electrode. The CILE was prepared by using N-hexylpyridinium hexafluorophosphate (HPPF(6)) as the binder with the graphite powder. The CTS-V(2)O(5)-MWCNTs/CILE was used as the basal electrode for the immobilization of the single-stranded DNA (ssDNA) probe. After the hybridization with the target ssDNA sequence, the electrochemical indicator of methylene blue (MB) was used to monitor the hybridization reaction. Experimental data indicated that the synergistic effect of nano-V(2)O(5) and MWCNTs increased the amounts of ssDNA adsorbed on the electrode surface and resulted in the corresponding increase of the electrochemical responses. This DNA biosensor combined the advantages such as the biocompatibility of V(2)O(5) nanobelt, the excellent electron transfer ability of MWCNTs, the good film-forming ability of CTS and the high conductivity of CILE. Under the optimal conditions differential pulse voltammetry (DPV) was used to record the electrochemical response of MB and the specific ssDNA sequence could be detected in the concentration range from 1.0x10(-11) to 1.0x10(-6) mol L(-1) with the detection limit as 1.76x10(-12) mol L(-1) (3sigma). The DNA biosensor showed good stability and discrimination ability to the one-base and three-base mismatched ssDNA sequence. The loop-mediated isothermal amplification (LAMP) product of Yersinia enterocolitica gene sequence in pork meat was detected by the proposed method with satisfactory result, suggesting that the CTS-V(2)O(5)-MWCNTs/CILE had the potential for the sensitive detection of specific gene sequence.