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、胃液分泌和神经元传递中发挥作用。为了实时监测肥大细胞、肠嗜铬细胞和组胺神经元等含组胺细胞的组胺释放，我们研制了一种基于重组组胺氧化酶的组胺微传感器。组胺传感器基于对辣根过氧化物酶和HAO催化的组胺产物过氧化氢的氧化还原反应产生的还原电流的安培测定。Hao是一种在全球节杆菌中发现的含铜/多酚的胺氧化酶，并已进行了测序。带有组氨酸标签的Hao在大肠杆菌中过表达，利用组氨酸亲和层析柱可以很容易地纯化。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。该传感器采用含辣根过氧化物酶、组胺氧化酶和银凝胶修饰的三种碳膜电极。当以2μL/分钟的流速吸入组胺标准溶液时，检测电流与组胺浓度呈线性关系，检测下限为11.3 nm。用20 ng/mlDNP-BSA作为抗原刺激大鼠嗜碱性白血病细胞(1×106)，组胺浓度为2.7μM。该传感器可用于肥大细胞等培养细胞组胺释放的分析。

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.