Cattaneo M V, Male K B, Luong J H
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
Biosens Bioelectron. 1992;7(8):569-74. doi: 10.1016/0956-5663(92)85008-x.
A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.
已开发出一种化学发光光纤生物传感器系统，用于测定在产生针对病毒表面抗原的单克隆抗体的杂交瘤细胞培养物中的谷氨酰胺。谷氨酰胺酶和谷氨酸氧化酶（GLO）分别通过戊二醛活化固定在氨丙基玻璃珠上，并装填在柱中。将两根分别装有固定化GLO和过氧化氢酶的独立柱子置于上游，以消除内源性谷氨酸。在铁氰化物存在下，鲁米诺与酶促反应释放的过氧化氢反应产生化学发光（CL）光信号，该信号通过光纤系统进行检测和定量。结合流动注射分析，可以几乎相同地处理样品，从而避免了重现CL信号的困难。在10^(-6)至10^(-3) M的范围内，CL响应与标准谷氨酰胺浓度之间存在极好的线性关系。包括进样和清洗在内，一次完整分析可在2分钟内完成。每个固定化酶柱至少可稳定进行300次重复分析而无任何活性损失。当该生物传感器系统用于测定哺乳动物细胞培养废液中的谷氨酰胺时，所得值与高效液相色谱法的值相比良好，从而验证了CL光纤系统的适用性。
