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
过氧化氢（hydrogen peroxide, H2O2）；样品基质：pH 7.0 磷酸盐缓冲液（PBS）
检测原理
该传感器基于Al3+指导的PAM自组装与酶直接电化学。Al3+与PAM水解羧基配位，在GC电极上形成有序三维树状多孔膜，将HRP和Pt/SWCNT异质结共同固定；Pt/SWCNT提供高导电通道，PAM多孔结构保持HRP天然构象并缩短电子转移路径。HRP血红素中心与电极发生直接电子转移，形成可逆氧化还原峰。当H2O2加入后，HRP-Fe(III)催化H2O2还原，电子经Pt/SWCNT和PAM网络快速传至电极，产生随H2O2浓度增大的阴极电流；高浓度下酶催化趋于饱和。该体系无外源标记，依靠酶电催化和纳米导电网络实现信号放大。
检测灵敏度
LOD: 0.08 μM (S/N = 3)；线性范围: 1–270 μM (1.0×10−6–2.7×10−4 M, R = 0.998)；灵敏度: 372 mA cm−2 M−1；表观Michaelis–Menten常数: 87 μM；ks: 14.94 ± 1.36 s−1；ΔEp: 约37 mV
效应效果
该传感器对H2O2响应快速稳定。在−0.28 V下，约4.6 s达到95%稳态电流，快于HRP/PAM电极的8.9 s。对120 μM H2O2连续7次测量RSD为2.6%，6个独立电极间RSD为3.4%；PBS浸泡4 h阴极峰电流下降<0.4%，4周保存保留96.0%初始响应。其LOD低于HRP/甲苯胺蓝-MWCNTs（1.7 μM）和溶胶-凝胶陶瓷-碳纳米管（12.89 μM）；线性范围宽于锆增强胶原三螺旋支架、明胶疏水离子液体凝胶和CeO2纳米管电极；灵敏度高于三维有序石墨化介孔碳传感器；KappM 87 μM小于570和1760 μM。作者认为可用于生物医学、食品和环境分析。
传感器的构成
- 基底/换能器电极：玻璃碳电极（GC electrode），抛光清洗后作为工作电极，提供电子转移界面。
- 纳米材料修饰层：铂/单壁碳纳米管异质结（Pt/SWCNT，Pt纳米颗粒约11 nm嵌入SWCNTs），由EDTA指导NaBH4还原H2PtCl6制备，提供导电通道并参与Al3+配位。
- 自组装固定化基质：聚丙烯酰胺（PAM，平均分子量约3×10^6）与Al3+（Al2(SO4)3，8.0 mM）配位形成三维树状多孔膜，固定酶和纳米材料并维持HRP天然构象。
- 识别元件/生物催化剂：辣根过氧化物酶（HRP，>250 U/mg），直接电化学活性中心并催化H2O2还原。
- 信号标记物：无外源标记，HRP血红素中心直接电子转移和电催化产生电流信号。
- 电化学体系：20 mM PBS（pH 7.0）作为电解质，Ag/AgCl参比电极和铂丝辅助电极构成三电极检测体系。
中文摘要
本文提出一种用于蛋白固定化和第三代生物传感器构建的通用新方法：Al3+指导聚丙烯酰胺（PAM）自组装形成有序三维树状结构，可便捷固定酶和纳米颗粒。作者首次采用EDTA指导合成策略制备铂/单壁碳纳米管（Pt/SWCNT）异质结纳米材料，并将其作为支撑基质，与Al3+指导的PAM自组装协同构建氧化还原蛋白固定化和生物传感平台。与近单层HRP/PAM膜电极相比，多层HRP/PAM/Pt/SWCNT膜电极在−0.22 V（vs. Ag/AgCl）处呈现显著增强的氧化还原峰。有序多层结构促进金属酶直接电子转移，表观异相电子转移速率常数ks为14.94±1.36 s−1，峰间分离约37 mV。该PAM/Pt/SWCNT基生物传感器对过氧化氢（H2O2）还原具有显著电催化活性，表观Michaelis–Menten常数为87 μM，线性范围为1–270 μM，检出限为0.08 μM（S/N=3），灵敏度为372 mA cm−2 M−1。结果表明，Al3+指导的HRP/PAM/Pt/SWCNT复合膜是氧化还原蛋白直接电化学及相关酶生物传感器的理想候选材料，有望用于生物医学、食品和环境分析检测。
英文摘要
A novel general methodology for protein immobilization and third-generation biosensor construction is demonstrated, which involves Al(3+)-directed polyacrylamide (PAM) self-assembly into an ordered dendriform structure, easily immobilizing enzymes and nanoparticles. Platinum/single-walled carbon nanotube (Pt/SWCNT) heterojunction nanomaterials were for the first time fabricated via an EDTA-directed synthesis strategy. The Pt/SWCNTs were employed as a supporting matrix to explore a novel immobilization and biosensing platform of redox proteins through cooperating Al(3+)-directed PAM self-assembly. Compared with the almost single-layer horseradish peroxidase (HRP)/PAM film electrode, multilayer HRP/PAM/Pt/SWCNT film electrode exhibited a pair of much stronger redox peaks at -0.22 V (vs. Ag/AgCl). Moreover, with advantages of the ordered multilayer HRP/PAM/Pt/SWCNT film, facilitated direct electron transfer of the metalloenzymes with an apparent heterogeneous electron transfer rate constant (k(s)) of 14.94+/-1.36 s(-1) and smaller peak-to-peak separation (DeltaE(p)) of about 37 mV was acquired on the PAM/Pt/SWCNT-based enzyme electrode. The PAM/Pt/SWCNT-based biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with a small apparent Michaelis-Menten constant (87 microM), wide linear range (1-270 microM), very low detection limit (0.08 microM, S/N=3), and high sensitivity (372 mA cm(-2) M(-1)). Together, these indicated that the Al(3+)-directed HRP/PAM/Pt/SWCNT film was one of ideal candidate materials for direct electrochemistry of redox proteins and the construction of the related enzyme biosensors, and may find potential applications in biomedical, food, and environmental analysis and detection.