Interferon gamma transcript detection on T cells based on magnetic actuation and multiplex double-tagging electrochemical genosensing

Interferon-γ is a proinflammatory cytokine, and its production is related with effective host defense against intracellular pathogens. Therefore, the level of interferon-γ is considered a good biomarker for intracellular infections. It is also useful for the assessment, treatment progression and follow-up of non-communicable diseases, including cancer and autoimmune disorders, among others. This work addresses the development of a novel interferon-γ release assay (IGRA) to evaluate the expression of interferon-γ transcripts as biomarker produced by isolated T cells, as a main advantage. The method sequentially combined three different types of magnetic separation, including the immunomagnetic separation of the T cells performed on antiCD3 modified magnetic particles, the retrotranscription and multiplex double-tagging PCR on polydT-modified magnetic particles and, finally, the electrochemical genosensing on streptavidin magnetic particles as a support. This approach is able to quantify the levels of cellular interferon-γ produced by as low as 150 T cells with outstanding analytical features. The detection of interferon-γ transcripts is performed from only 100 μL of whole blood which can be potentially obtained by fingerprick, demonstrating a further clear advantage to be considered as a promising strategy for the quantification of this important biomarker in several clinical applications.