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Quenching of fluorescence caused by Graphene Oxide as an immunosensing platform in a microwell plate format
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1  Biophotonic Nanosensors Laboratory, Research Center in Optics (registering DOI)

Nowadays immunoassays are used to detect chemical or biological species; therefore, they are an essential tool in a wide range of applications such as drug development, clinical diagnostics, environmental monitoring or food quality control [1]. However, conventional immunoassays, such as ELISA (Enzyme-Linked ImmunoSorbent Assay), require several procedures such as blocking, separations and washing steps. Thus, it takes at least 6 hours to get the respective results. Besides, it involves two antibodies and a sensing surface attaching and labelling the biochemical target (analyte). Fluorescence Resonance Energy Transfer (FRET) is a very useful phenomena to improve immunosensing sensitivity and avoid cumbersome procedures due to its simplicity. Graphene and its derivatives have been used as acceptors in FRET due to their wide absorption spectra, which make them outstanding quenchers of fluorescence. With this in mind, we developed a novel and single-step biosensing platform based on fluorescence quenching caused by graphene oxide, which was used for the detection of two analytes: H-IgG (which is a type and also the most common antibody found in human blood circulation) and PSA (prostate specific antigen). A single antibody conjugated with a fluorophore (FITC for H-IgG detection and quantum dots for PSA detection) is used in the capture and detection processes. When the analyte and the antibody (conjugated with the fluorophore) are added, a kinetic analysis is performed throughout 2 hours with real-time interrogation of the respective fluorescence intensity, observing that the higher analyte concentration, the less quenching of fluorescence of the immunosensing probe (antibody-fluorophore immunocomplex) due to the low affinity and the relatively long distance between GOµWs (microwells plate coated with Graphene Oxide) and immunosensing probe [2].


[1] In Introduction to Biophotonics, John Wiley & Sons, Ltd, 2004, pp. 311–356.

[2] E. Ortiz-Riaño, M. Avila-Huerta, D. Mancera-Zapata, E. Morales-Narváez, Biosensors and Bioelectronics 2020, 165, 112319.

Keywords: FRET; fluorescence; immunoassays; graphene; single-step biosensing platform.