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Bioassay engineering: A combined label-free and fluorescence approach to optimize HER2 detection in complex biological media

: Sinibaldi, A.; Doricchi, A.; Pileri, T.; Allegretti, M.; Danz, N.; Munzert, P.; Giordani, E.; Giacomini, P.; Michelotti, F.


Analytical and bioanalytical chemistry 412 (2020), No.14, pp.3509-3517
ISSN: 1618-2642 (Print)
ISSN: 1618-2650 (Online)
Journal Article
Fraunhofer IOF ()

We report on the combined label-free/fluorescence use of one-dimensional photonic crystals to optimize cancer biomarker detection in complex biological media. The optimization of the assay working parameters permits us to maximize the final response of the biosensor. The detection approach utilizes a sandwich assay, in which one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies in order to guarantee high specificity during biological recognition. The multiple outcomes generated by such optimization experiments permitted us to determine the effective capture efficiency and the repeatability of the immobilization process, which was estimated to be close to 5%. By exploiting the resolution of the fluorescence operation mode, we studied non-specific interactions in different blocking agents, different analyte diluting buffers, and diverse concentrations of the detection antibody. As a clinically relevant biomarker, we selected the trans-membrane receptor tyrosine kinase HER2. HER2 regulates a variety of cell proliferation, growth, and differentiation pathways and its over-expression occurs in approximately 20–30% of breast cancer worldwide. As a final application, we transferred all the optimized working parameters to HER2 cancer biomarker assays in a complex biological environment. The label-free and fluorescence results obtained by analyzing MCF-7 (HER2 low positive) and 32D (HER2 negative) cell lysates demonstrate that we can successfully discriminate the two lysates.