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Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications ‐ bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we usedGaussialuciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV‐1 DNA and IFN‐γ. We demonstrated that the incubation of Gluc conjugated HIV‐1 and IFN‐γ oligonucleotide probes with GO provided for monitoring of probe‐target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN‐γ and HIV‐1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN‐γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter.

The ability to monitor types, concentrations, and activities of different biomolecules is essential to obtain information about the molecular processes within cells. Successful monitoring requires a sensitive and selective tool that can respond to these molecular changes. Molecular aptamer beacon (MAB) is a molecular imaging and detection tool that enables visualization of small or large molecules by combining the selectivity and sensitivity of molecular beacon and aptamer technologies. MAB design leverages structure switching and specific recognition to yield an optical on/off switch in the presence of the target. Various donor–quencher pairs such as fluorescent dyes, quantum dots, carbon‐based materials, and metallic nanoparticles have been employed in the design of MABs. In this work, the diverse biomedical applications of MAB technology are focused on. Different conjugation strategies for the energy donor–acceptor pairs are addressed, and the overall sensitivities of each detection system are discussed. The future potential of this technology in the fields of biomedical research and diagnostics is also highlighted.