An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis.
An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis.
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DOI：
10.1039/a907709j
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
V. C. Tsafack;C. Marquette;B. Leca;L. Blum
中科院分区：
文献类型：
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作者：
V. C. Tsafack;C. Marquette;B. Leca;L. Blum
建立了一种基于鲁米诺电化学发光（ECL）的光纤生物传感器，并将其与流动注射分析（FIA）系统相结合，用于胆碱的检测。鲁米诺的电化学发光是由玻碳电极在+425 mV极化相对于铂伪参比电极。胆碱氧化酶（Chx）被共价固定在聚酰胺（ABC型）或UltraBind预活化膜上，或通过物理包埋在光交联聚乙烯醇聚合物（PVA-SbQ）中，或在弱阴离子交换剂DEAE（二乙基氨基乙基）Sepharose上吸附后固定。影响FIA生物传感器响应的反应条件和物理化学参数的优化表明，胆碱生物传感器在含有30 mM KCl和1.5 mM MgCl 2的30 mM veronal缓冲液中表现出最佳性能，pH 9。使用0.5 ml min-1的流速，使胆碱的测量由基于膜的ECL生物传感器在8或5分钟内，分别与ABC或UltraBind膜，而测量只需要3分钟的DEAE-PVA系统。为了进行比较，胆碱的检测与Chx固定使用四种不同的支持。DEAE-PVA-Chx传感层获得了最佳性能，其允许10 pmol的检测限，而ABC、UltraBind和PVA系统的检测限分别为300 pmol、75 pmol和220 pmol。基于DEAE的系统也表现出良好的操作稳定性，因为可以进行160次重复测量3 nmol胆碱，RSD为4.5%，而在最佳条件下的稳定性为45次测定。
A fibre optic biosensor based on luminol electrochemiluminescence (ECL) integrated in a flow injection analysis (FIA) system was developed for the detection of choline. The electrochemiluminescence of luminol was generated by a glassy carbon electrode polarised at +425 mV vs. a platinum pseudo-reference electrode. Choline oxidase (Chx) was immobilised either covalently on polyamide (ABC type) or on UltraBind preactivated membranes, or by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) alone or after absorption on a weak anion exchanger, DEAE (diethylaminoethyl) Sepharose. The optimisation of the reaction conditions and physicochemical parameters influencing the FIA biosensor response demonstrated that the choline biosensor exhibited the best performances in a 30 mM veronal buffer containing 30 mM KCl and 1.5 mM MgCl2, at pH 9. The use of a 0.5 ml min-1 flow rate enabled the measurement of choline by the membrane-based ECL biosensors in 8 or 5 min, with ABC or UltraBind membranes, respectively, whereas the measurement required only 3 min with the DEAE-PVA system. For comparison, the detection of choline was performed with Chx immobilised using the four different supports. The best performances were obtained with the DEAE-PVA-Chx sensing layer, which allowed a detection limit of 10 pmol, whereas with the ABC, the UltraBind and the PVA systems, the detection limits were 300 pmol, 75 pmol and 220 pmol, respectively. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with an RSD of 4.5% whereas the stability under the best conditions was 45 assays with the other supports.