Departamento de Química Analítica, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
Anal Bioanal Chem. 2012 Apr;402(10):3211-8. doi: 10.1007/s00216-011-5701-0. Epub 2012 Feb 14.
This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 μg mL(-1), respectively, and a dynamic range from 0.52 to 61.05 μg mL(-1) TFP (n = 5, correlation coefficient 0.998). The response time (t(100)) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%.
本文描述了一种新型在线生物传感器的研制，该传感器基于荧光标记的人钙调蛋白（CaM），即 hCaM M124C-mBBr，固定在可控孔玻璃（CPG）上，用于分析三氟拉嗪（TFP）；一种人尿样中的吩噻嗪类药物。该装置通过将 hCaM M124C-mBBr-CPG 填充在与监测系统相连的连续流动微池中实现自动化，该监测系统由分叉光纤耦合到荧光分光光度计组成。在线生物传感器的操作参数（流速、样品注入量、载液和缓冲液 pH 值）进行了研究和优化。在最佳条件下，该生物传感器的检测限和定量限分别为 0.24 和 0.52μg·mL-1，检测范围为 0.5261.05μg·mL-1 TFP（n=5，相关系数 0.998）。响应时间（t（100））短于 42s（恢复时间<4.5min），在线性响应范围内，TFP 测量的重现性和重复性低于 1.4%和 2.7%。该装置成功应用于加标人尿样中 TFP 的分析，回收率在 97%101%之间，相对标准偏差（RSD）低于 5.9%。
