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
尿素（urea）；样品基质：Tris 缓冲液（pH 8.1）/水溶液
检测原理
尿素扩散进入固定化脲酶膜，脲酶催化尿素水解生成氨/铵离子和碳酸氢根。氨/铵本身不具直接电活性，无法被安培电极直接检测；负载在膜孔中的铑纳米颗粒在碱性 Tris 缓冲液（pH 8.1）中催化氨/铵在铂工作电极表面氧化为氮气。该催化氧化在 0.8 V 恒电位下产生阳极电流，铑纳米颗粒同时改善酶活性位点与电极间的电子传递。随着尿素浓度增加，酶促反应生成的氨量增加，催化氧化电流随之增大，在 1.6–8.2 mM 范围内呈线性响应。传感器通过记录稳态安培电流实现尿素定量。
检测灵敏度
LOD: 0.5 mM (S/N = 3)；线性范围: 1.6–8.2 mM；灵敏度: 3.1927 μA mM−1 cm−2；R^2 = 0.998；校准方程: I (μA) = 3.1927x − 0.0193
效应效果
传感器响应迅速，约 20 s 达到稳态电流。同一传感器在 1.6 mM 尿素下连续 5 次测定的相对标准偏差为 5.9%；使用 10 天后最大响应保持 86.8%。固定化脲酶在 4°C 储存 50 天后保留 93% 活性，而游离脲酶仅保留 50%；70°C 热失活 180 min 后仍保留约 80% 活性，游离酶完全失活；连续 10 次操作后活性损失约 9%。作者指出，不对称 AN 膜孔可将酶截留在非选择性侧孔内，减少酶流失并降低电化学干扰。其可更换酶膜设计降低电极更换成本，适用于低浓度尿素快速检测。
传感器的构成
- 换能器电极：铂工作电极（Pt working electrode, d=10 mm），提供电子传递并输出安培电流
- 膜基底：丙烯腈–甲基丙烯酸甲酯–乙烯磺酸钠共聚物膜（AN copolymer membrane），多孔载体，截留酶与纳米颗粒
- 化学改性层：NaOH/乙二胺（EDA）处理及戊二醛（GA）接枝，引入氨基并用于交联
- 壳聚糖修饰层：壳聚糖（chitosan, CHI, 1.0%），提供氨基、亲水性和生物相容性
- 催化/导电纳米层：铑纳米颗粒（Rh NPs, 5% on activated charcoal），催化氨氧化并增强电子传递
- 识别元件：脲酶（urease, Ure, E.C. 3.5.1.5），催化尿素水解生成氨/铵
- 交联剂：戊二醛（glutaraldehyde, GA），交联固定脲酶并稳定膜表面
中文摘要
本研究通过在不同浓度（0.15%、0.3%、0.5%、1.0%、1.5%）壳聚糖化学接枝及负载铑纳米颗粒（5% 负载于活性炭）到预先化学改性的丙烯腈（AN）共聚物膜中，获得一种用于脲酶固定化的新型基质。对壳聚糖改性膜的基本特性进行了研究，扫描电镜分析表明不同改性膜的表面形貌发生显著变化。测定了固定化酶的蛋白结合量和相对活性，结果显示负载于 1.0% 壳聚糖修饰并含铑纳米颗粒的 AN 共聚物膜上的脲酶相对活性最高，约为 77.44%。进一步确定了该优化膜上固定化脲酶的最适 pH、最适温度、热稳定性、储存稳定性和操作稳定性。将制备的酶膜用于构建尿素安培生物传感器，并研究其基本安培特性。在 1.6–23 mM 尿素范围内获得校准曲线，其中 1.6–8.2 mM 呈线性；传感器灵敏度为 3.1927 μA mM−1 cm−2，相关系数为 0.998；信噪比为 3 时尿素检出限为 0.5 mM。传感器使用 10 天后，对尿素的最大响应仍保持 86.8%。
英文摘要
A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.