Immunoassay-Immunosensors Laboratory, Institute of Radioisotopes and Radiodiagnostic Products, N.C.S.R. Demokritos, Aghia Paraskevi, Athens, 15310, Greece.
Anal Bioanal Chem. 2010 Feb;396(3):1187-96. doi: 10.1007/s00216-009-3278-7. Epub 2009 Nov 21.
The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin-horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction.
急性心肌梗死的早期诊断需要在发病后短时间内测定血清中的几种标志物。最广泛使用的标志物是肌酸激酶同工酶 MB（CK-MB）和心肌肌钙蛋白 I（cTnI）。在本工作中，展示了一种用于快速和高灵敏度同时测定血清样品中这些标志物的毛细管波导荧光免疫传感器。双分析物免疫传感器是使用内部用超薄膜聚二甲基硅氧烷（PDMS）修饰的玻璃毛细管实现的，通过在离散的分析物特异性抗体带上创建。然后将毛细管充满样品和生物素化检测抗体的混合物，接着与链霉亲和素-辣根过氧化物酶反应，并与荧光标记的酪胺衍生物孵育，以将荧光标记物累积到免疫反应带上。当用激光束扫描毛细管时，部分发射荧光被捕获并通过毛细管壁波导到放置在其一端的光电倍增管。与使用传统荧光标记物 R-藻红蛋白在 65 分钟测定中获得的 0.8 和 0.6 ng/mL 相比，使用酪胺信号放大的方法分别使 cTnI 和 CK-MB 的检测限降低至 0.2 和 0.5 ng/mL，总测定时间为 30 分钟。此外，所提出的免疫传感器提供了准确和可重复的测量（内测定和间测定的变异系数低于 10%），并且在血清样品中确定的值与商用酶免疫测定法获得的值非常吻合。因此，所提出的毛细管波导荧光免疫传感器具有快速可靠诊断急性心肌梗死所需的所有特征。
