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
电化学生物传感器
检测对象
Fenton试剂（Fe2+/H2O2）及其产生的羟基自由基（·OH）诱导的酪氨酸氧化（tyrosine oxidation，L-多巴）；样品基质：纯水孵育体系（电极表面多肽模型）
检测原理
传感器以ITO/PDDA/poly(glu,tyr)(4:1)多层膜为识别界面，多肽中的酪氨酸残基作为响应位点。Fenton试剂Fe2+/H2O2在孵育体系中发生Fenton反应生成羟基自由基（·OH），·OH攻击酪氨酸酚羟基，将其氧化为L-多巴。L-多巴的儿茶酚基团可与Fenton反应中生成的Fe(III)形成Fe(III)-L-多巴螯合物，使儿茶酚基团电活性降低；同时Os(bpy)3^2+作为电催化剂介导未氧化酪氨酸及L-多巴的氧化，产生阳极电流。随着Fe2+或H2O2浓度升高，酪氨酸氧化和Fe(III)螯合增强，阳极电流下降。DFO竞争结合Fe(III)可部分恢复电流，XPS证明多肽主链未断裂，说明电流下降主要源于酪氨酸氧化及螯合抑制。
检测灵敏度
LOD: 10 μM Fe2+ 或 H2O2（原文表述为最低可检测Fenton试剂浓度）
效应效果
该传感器对Fenton试剂具有明显响应：单独H2O2对多肽电流几乎无影响，单独Fe2+仅使信号缓慢下降，30 min后降至约60%；FeSO4（1 mM）/H2O2（4 mM）孵育后电流降至初始信号的30%。加入铁螯合剂DFO后电流回升，支持Fe(III)-L-多巴螯合机制。多肽修饰电极可稳定保存约2周，厌氧条件下更久；三次重复实验误差多在1%–15%。最低可检测Fe2+或H2O2浓度为10 μM，接近体内水平。作者认为相比传统方法，该法灵敏、快速、成本低，可用于Fenton反应诱导的蛋白氧化损伤检测。
传感器的构成
- 基底/换能器电极：氧化铟锡（ITO）导电玻璃电极，工作面积25 mm2，提供导电基底与电子转导
- 聚合物修饰层：聚二甲基二烯丙基氯化铵（PDDA）正电荷聚合物，通过静电吸附构建层状结构并固定多肽
- 识别/响应元件：poly(glu, tyr)(4:1)钠盐多肽，含酪氨酸残基，作为Fenton反应氧化损伤的模型识别元件
- 信号报告物：三(2,2'-联吡啶)锇(II) Os(bpy)3^2+，介导酪氨酸及L-多巴的电化学氧化，产生阳极电流
- 支持电解质：20 mM磷酸盐缓冲液（PB，pH 7.4），提供离子导电环境
中文摘要
本文报道了一种简单、灵敏的电化学生物传感器，用于检测Fenton反应（Fe2+/H2O2）产生的羟基自由基诱导的酪氨酸氧化。将poly(glu, tyr)(4:1)多肽通过层层自组装技术固定在氧化铟锡（ITO）电极表面，并以三(2,2'-联吡啶)锇(II) Os(bpy)3^2+介导的酪氨酸氧化电流作为信号报告。结果表明，多肽与Fenton试剂孵育后电化学信号显著下降。进一步发现，酪氨酸氧化产物L-多巴可能与Fe(III)形成螯合物，从而抑制L-多巴的电化学氧化并导致电流响应降低。作者认为多肽损伤包含两步过程，并符合二级反应动力学。X射线光电子能谱定量测定了多肽修饰电极表面的氮元素含量，排除了信号下降由多肽主链断裂引起的可能。该传感器可检测的Fenton试剂最低浓度为10 μM Fe2+或H2O2，接近体内水平，提示其可用于检测Fenton反应诱导的蛋白氧化损伤。
英文摘要
A simple and sensitive electrochemical biosensor was used to detect tyrosine oxidation induced by hydroxyl radicals generated by Fenton reaction (Fe(2+)/H(2)O(2)). Poly(glu, tyr) (4:1) peptides were immobilized on indium tin oxide (ITO) electrode surface via layer-by-layer assembly technique, and Os(bpy)(3)(2+)-mediated tyrosine oxidation current was employed as the signal reporter of the biosensor. It was found that the electrochemical signal of the peptide decreased markedly after incubation with Fenton reagents. Interestingly, L-dopa, the oxidation product of tyrosine, was likely to form complexes with Fe(III), which could suppress the electro-oxidation of L-dopa and resulted in decrease of current response. Our results indicate that the peptide damage involved two steps and was a second-order reaction. X-ray photoelectron spectroscopy was used to quantitatively determine nitrogen elemental percentage on peptide-coated electrode surface, which eliminated the possibility that signal decrease was caused by peptide backbone cleavage. Moreover, the lowest concentration of Fenton reagents that could be detected was 10 μM Fe(2+) or H(2)O(2), similar to the level in vivo. We suggest that the biosensor can be used to detect protein damage induced by Fenton reaction.