Departamento de Química Analítica, Instituto Químico para la Energía y el Medioambiente (IQUEMA), Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie, Córdoba, E-14071, España.
Mikrochim Acta. 2023 May 11;190(6):209. doi: 10.1007/s00604-023-05786-z.
A stopped-flow microfluidic fluorimetric biosensor to monitor alkaline phosphatase (ALP) activity and evaluate the potential inhibitors has been developed, integrating a magnetically retained enzyme microreactor (MREµR) in the reaction/detection zone of the microfluidic chip. The integration supposed the alignment of the MREµR at the sample compartment of a conventional spectrofluorometer using a 3D-printed device. The analytical signal is based on the fluorescence decrease in the signal obtained in the dephosphorylation reaction of the substrate 4-methylumbelliferone phosphate (4-MUP) by the retained ALP-MNPs in an alkaline medium caused by sulfonamides. The excitation and emission wavelengths to monitor the reaction were 363 and 444 nm, respectively. Three sulfonamides, acetazolamide, furosemide, and sulfasalazine, have been used as model analytes. The front-face operating mode of the spectrofluorometer was used to acquire the instrumental signals. The influence of the rotation angle of the microfluidic device on the efficiency of the signal collection has also been studied, obtaining the signals with greater intensity at 75° from the excitation beam. The dynamic range of the calibration graph was 16.81-1111.22 µg mL, expressed as sulfonamide concentration, with a limit of detection of 5.04 µg mL (R = 0.9989, n = 10, r = 3) for acetazolamide. The method was applied to determine sulfonamide residues in tap water and milk samples, with 88.9-98.7% recovery values. The results have been compared with those obtained using a commercial device connected to the spectrofluorometer, getting faster reaction kinetics.
一种用于监测碱性磷酸酶（ALP）活性并评估潜在抑制剂的停流微流荧光生物传感器已经被开发出来，该传感器将磁性保留酶微反应器（MREµR）集成到微流控芯片的反应/检测区域中。这种集成需要使用 3D 打印设备将 MREµR 对准传统分光光度计的样品室。分析信号基于在碱性介质中保留的 ALP-MNPs 对底物 4-甲基伞形酮磷酸盐（4-MUP）的去磷酸化反应导致的荧光强度降低，该信号是通过磺酰胺获得的。用于监测反应的激发和发射波长分别为 363nm 和 444nm。三种磺酰胺，即乙酰唑胺、呋塞米和柳氮磺胺吡啶，被用作模型分析物。使用分光光度计的前向操作模式获取仪器信号。还研究了微流控设备的旋转角度对信号采集效率的影响，从激发光束获得了强度更大的信号。校准曲线的动态范围为 16.81-1111.22μgmL，以磺酰胺浓度表示，乙酰唑胺的检测限为 5.04μgmL（R=0.9989，n=10，r=3）。该方法已应用于测定自来水中和牛奶样品中的磺胺残留，回收率为 88.9-98.7%。将结果与使用连接到分光光度计的商业设备获得的结果进行了比较，得到了更快的反应动力学。
