Real-time monitoring of histamine released from a single histamine neuron with a histamine microsensor using recombinant histamine oxidase

组胺是一种生物活性胺，在过敏反应、胃分泌和神经传递中起作用。为了实时监测含有组胺的细胞（如肥大细胞、肠嗜铬细胞和组胺神经元）释放的组胺，我们利用重组组胺氧化酶（HAO）开发了一种组胺微传感器。组胺传感器是基于用电流法测定辣根过氧化物酶与过氧化氢（HAO催化组胺的产物之一）氧化还原反应产生的还原电流。HAO是一种在球形节杆菌中发现的含铜/托帕醌的胺氧化酶，已经测序。用组氨酸亲和柱纯化了在大肠杆菌中过表达的带有组氨酸标签的HAO。该酶对组胺的催化活性高于二胺（如腐胺和尸胺）或n -甲基组胺。该传感器采用含有辣根过氧化物酶、组胺氧化酶和银的锇-聚乙烯吡啶凝胶修饰的三个碳膜电极。以2 μl/min的流速抽吸组胺标准溶液时，检测电流与组胺浓度成正比，检测下限为11.3 nM。以20 ng/ml DNP-BSA作为抗原刺激大鼠嗜碱性白血病细胞（1×10^6），组胺浓度为2.7μM。这些结果表明，微传感器可用于分析培养细胞（如肥大细胞）的组胺释放。

Histamine, a bioactive amine, plays roles in allergic reaction, gastric secretion and neuronal transmission. For real-time monitoring of histamine release from histamine containing cells such as mast cells, enterochromaffin-like cells and histamine neurons, we developed a histamine microsensor using recombinant histamine oxidase(HAO). The histamine sensor is based on the determination of the reducing current amperometrically resulted from the redox reaction with horseradish peroxidase and hydrogen peroxide which is one of the products of histamine catalyzed by HAO. HAO is a copper/topa quinone-containing amine oxidase found in Arthrobacter globiformis and has already sequenced. HAO with a histidine tag, which was overexpressed in E.coli, was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g.putrescine and cadaverine) or N-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpiridine-based gel containing horseradish peroxidase, histamine oxidase and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 μl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1×10^6) were stimulated by 20 ng/ml of DNP-BSA as an antigen, the histamine concentrations were 2.7μM.These results indicate that the microsensor is useful for the analysis of histamine release from cultured cells such as mast cells.