Dual-cardiac marker capillary waveguide fluoroimmunosensor based on tyramide signal amplification

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.