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Porphyrins and metalloporphyrins are promising new protein labels that can be detected using multiple techniques; improving the reliability of the analysis and broadening the range of the linear response. Here, we investigate the potential of 5,10,15,20-tetraphenyl-21H,23H-porphyrin (Tpp) as a hybrid protein label. The electrochemical and optical properties of porphyrin conjugated with bovine serum albumin (BSA), chicken egg albumin (CEA) and immunoglobulin G (IgG) were determined and optimal conditions for Tpp-protein conjugation established. Model conjugates of carboxylated Tpp with BSA and short peptides were characterized using differential pulse voltammetry, UV–Vis spectrophotometry and spectrofluorimetry. These results reveal that Tpp is a promising molecule to be used in a triple detection protein labelling system.

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Herein, the conjugation of carboxylated tetraphenylporphyrin or its derivative containing manganese cation and model protein — immunoglobulin G is presented. The obtained IgG–cTpp and IgG–Mn-cTpp conjugates were subsequently used for model immunoassays construction. The IgG–cTpp formation was confirmed using size-exclusion chromatography. Thanks to the unique properties of applied labels the assay analysis was carried out with both spectrophotometric and spectrofluorimetric detection. The assays were performed creating semi-quantitative detection system using 96-well plates. The incubation time, ensuring full saturation of the surface with secondary antibodies was also optimized. Moreover, in the case of IgG–Mn-cTpp conjugates we present the possibility of both direct and indirect determination of the label, the latter based on the catalytic activity of Mn-cTpp, which allows for amplification of the measured signal. We proved that both cTpp and Mn-cTpp may be successfully used for protein labeling and serve as universal tracers for various formats of affinity assays and sensors.