组成图示
示意图生成中
传感器类型
全细胞生物传感器
检测对象
酚类化合物（phenolic compounds，包括phenol、catechol、hydroquinone、benzoic acid、1,2-dihydroxynaphthalene、L-tyrosine、L-DOPA、L-syringic acid、methyl-parathion）；样品基质：合成废水（synthetic wastewater）及MSM缓冲液
检测原理
固定于电极表面的铜绿假单胞菌全细胞作为识别元件，酚类化合物进入细胞后被酚羟化酶等代谢途径氧化降解。该呼吸过程消耗溶解氧，使电极附近氧浓度下降。Clark氧电极或厚膜金工作电极在-700 mV（vs Ag/AgCl）下对氧进行安培检测，氧浓度降低导致电流下降。底物浓度越高，细胞耗氧速率越大，电流下降幅度越大，从而实现定量检测。酚适应细胞因诱导产生酚降解酶系，对酚及某些酚类底物具有更高响应和选择性；该传感器无外源标记，也未采用HCR、RCA等额外放大策略，信号直接来自细胞呼吸耗氧。
检测灵敏度
线性范围: Phenol (control cells) 0.10–1.00 mM；slope 16.54 mA.M-1；R^2 0.9986；Catechol 0.50–5.00 mM；slope 5.07；R^2 0.9971；Hydroquinone 0.25–1.00 mM；slope 15.20；R^2 0.9972；Benzoic acid 0.10–0.75 mM；slope 15.82；R^2 0.9981；1,2-Dihydroxynaphthalene 0.25–2.50 mM；slope 8.01；R^2 0.9992；Phenol (adapted cells) 0.50–6.00 mM；slope 5.03；R^2 0.9995；L-Tyrosine 0.02–0.20 mM；slope 87.08；R^2 0.9988；L-Syringic acid 0.02–0.20 mM；slope 37.01；R^2 0.9852；L-DOPA 0.02–0.20 mM；slope 61.88；R^2 0.9989；Methyl-parathion 0.30–2.50 mM；slope 3.64；R^2 0.9917。
效应效果
适应细胞与对照细胞对不同酚类底物响应不同：适应细胞对L-DOPA、L-tyrosine、L-syringic acid、resorcin、picric acid和methyl-parathion有响应，对照细胞无响应，表明适应过程改变底物特异性。厚膜传感器在MSM、28°C下6 h内25次测量无响应下降，之后信号降低，需每日制备。重现性：适应细胞2.0 mM phenol（n=7）S.D.±0.09 mM，C.V.4.4%；对照细胞0.5 mM phenol（n=7）S.D.±0.02 mM，C.V.3.9%。两种合成废水中0.25、0.50、1.0、2.0 mM酚检测值分别为0.24±0.01、0.52±0.02、1.01±0.02、1.98±0.05 mM（适应）和0.25±0.01、0.51±0.03、1.01±0.02、2.02±0.03 mM（对照），无基质效应，无需前处理。作者认为其成本低、易制备、无需酶纯化，可用于废水酚快速检测。
传感器的构成
- 换能器电极：商用Clark氧电极（Pt工作电极，Teflon膜）或陶瓷厚膜传感器（Au工作电极、Ag/AgCl参比电极、Pt辅助电极），用于检测氧浓度变化
- 固定层：225 bloom gelatin（明胶）与细胞混合后涂覆于电极表面，干燥形成生物活性层
- 识别元件：Pseudomonas putida DSM 50026全细胞（酚适应细胞或葡萄糖对照细胞），通过呼吸代谢酚类化合物消耗O2
- 交联层：2.5% glutaraldehyde（戊二醛）在50 mM phosphate buffer（pH 7.5）中交联明胶，固定细胞并保护活性
- 工作介质：MSM（mineral salts medium，pH 6.9）作为工作缓冲液，维持细胞代谢活性
- 信号标记物：无外源标记，以细胞呼吸消耗O2引起的电流变化作为信号
中文摘要
本研究开发了一种基于细菌全细胞的安培型生物传感器，用于酚类化合物的测定，其测量基于细胞的呼吸活性。为此，选用著名的酚降解菌铜绿假单胞菌（Pseudomonas putida DSM 50026）作为生物识别元件。细胞在酚作为唯一有机碳源的培养基中生长，获得酚适应细胞；同时，以葡萄糖为主要碳源培养的细菌也用于制备另一类生物传感器，以便比较其对不同外源化合物的响应和特异性。研究采用商用氧电极作为换能器，分别测试诱导细胞和非诱导细胞的传感器响应。结果表明，适应步骤可获得具有不同底物特异性的生物传感器装置。此外，将铜绿假单胞菌用明胶膜固定于金厚膜工作电极表面，明胶膜经戊二醛交联。传感器对不同酚类物质进行了校准，并在合成废水样品中完成了酚的检测。
英文摘要
Amperometric biosensors using bacterial cells were developed for the determination of phenolic compounds and the measurement was based on the respiratory activity of the cells. For this purpose, Pseudomonas putida DSM 50026 which is one of the well-known phenol degrading organisms, was used as a biological component. The cells were grown in the presence of phenol as the sole source of organic carbon. As well as phenol adapted cells, the bacterium which used the glucose as the major carbon source, was also used to obtain another type of biosensor for the comparison of the responses and specificities towards different xenobiotics. The commercial oxygen electrode was used as a transducer to test the sensor responses for both induced and non-induced cells. Our results showed that the adaptation step enable us to obtain biosensor devices with different substrate specificity. Moreover, P. putida was immobilized on the surface of thick film working electrodes made of gold by using gelatin membrane cross-linked with glutaraldehyde. The biosensors were calibrated for different phenolic substances. Furthermore, phenol detection was performed in synthetic wastewater samples.