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
金黄色葡萄球菌（Staphylococcus aureus, S. aureus ATCC25923），样品基质：PBS细菌悬液（磷酸盐缓冲液）
检测原理
该传感器为无标记电化学阻抗免疫传感器。金电极表面先形成MPA自组装单分子层，经NHS/EDC活化后共价固定抗S. aureus抗体，并用乙醇胺封闭。当PBS中的S. aureus与抗体特异性结合后，电极表面生物层厚度增加，界面疏水性和电荷屏障增强，阻碍[Fe(CN)6]^3-/4-在Au表面发生电子转移。EIS施加小振幅正弦扰动，Nyquist图中半圆直径增大，电荷转移电阻Rct升高。通过Rs-Rct-CPE-Warburg等效电路拟合，Rct增量与细菌浓度对数值在10–10^6 CFU/mL内线性相关，灵敏度为127 Ω/decade。
检测灵敏度
LOD: 10 CFU/mL；线性范围: 10–10^6 CFU/mL；灵敏度: 127 Ω/decade；相关系数: 0.994
效应效果
总体来看，该传感器对金黄色葡萄球菌具有良好选择性，对大肠杆菌和表皮葡萄球菌无显著响应；正负响应比显示对大肠杆菌的干扰低于表皮葡萄球菌。四个独立制备传感器的平均标准偏差为8%，重现性良好。与文献中阻抗、安培和SPR方法相比，作者认为其检出限更低、动态范围更宽，适用于病原菌快速筛查。传感器不可再生，抗体无法用于后续检测，这是主要局限。
传感器的构成
- 基底/换能器：金电极（Au electrode），作为工作电极和生物识别界面载体
- 自组装单分子层：3-巯基丙酸（MPA）SAMs，通过硫醇在金表面化学吸附并提供末端羧基
- 活化层：N-羟基琥珀酰亚胺/碳二亚胺（NHS/EDC），活化MPA末端羧基形成活性酯
- 识别元件：抗金黄色葡萄球菌多克隆抗体（anti-S. aureus antibodies），与活化羧基共价结合并识别细菌
- 封闭层：乙醇胺（ethanolamine），封闭剩余酸性基团以减少非特异性吸附
- 氧化还原探针：铁氰化钾/亚铁氰化钾（K3Fe(CN)6/K4Fe(CN)6，Fe(CN)6^3-/4-），在PBS中提供电子转移信号
- 检测体系：磷酸盐缓冲液（PBS）与三电极体系（Au工作电极、Pt辅助电极、SCE参比电极），用于CV和EIS测量
中文摘要
病原菌检测仍是生物防御、院内感染和食品安全领域的挑战。本研究旨在开发一种简便的电化学免疫传感器，用于检测致病性金黄色葡萄球菌ATCC25923。该传感器通过3-巯基丙酸（MPA）自组装单分子层（SAMs）固定抗金黄色葡萄球菌抗体而构建。将基底浸入含SAMs的有机溶剂中，使分子自组装层与金表面共价结合。采用循环伏安法（CV）和电化学阻抗谱（EIS）对免疫传感器功能化过程进行表征，分析在含铁氰化/亚铁氰化氧化还原探针的磷酸盐缓冲液中进行。EIS用于抗体-细胞结合亲和实验。结果表明，电荷转移电阻（RCT）增量与金黄色葡萄球菌浓度的对数值在10–10^6 CFU/mL范围内呈线性关系，检出限为10 CFU/mL，重现性为8%。与大肠杆菌和表皮葡萄球菌相比，该传感器表现出良好选择性，证明其对金黄色葡萄球菌具有特异性。
英文摘要
The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs) of 3-Mercaptopropionic acid (MPA). These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (RCT) and the logarithmic value of S. aureus concentration was observed between 10 and 106 CFU/mL. The limit of detection (LOD) was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus.