A novel ratiometric biosensor based on germanene nanosheets for rapid and sensitive BRCA1 detection in differentiating T cells

Abstract

The BRCA1 gene is essential for the developmental regulation and function of T lymphocytes, yet its mRNA expression during T-cell differentiation remains unclear due to the lack of effective in-situ monitoring tools. To address this, we developed a ratiometric fluorescent nanosensor based on Förster resonance energy transfer (FRET) for reliable quantification of BRCA1 mRNA in living CD8+ T cells. The sensor comprises a fluorescein-labeled DNA probe (FAMcDNA) assembled with Triton X-100-modified methylgermanene nanosheets (GeT), forming an efficient FRET pair. In the absence of the target, FAMcDNA adsorbs onto the GeT surface, resulting in a FRET effect that quenches FAM fluorescence and enhances GeT emission by 1.63-fold. Upon specific hybridization with BRCA1 mRNA, the probe detaches, disrupting the FRET process and causing a quantitative ratiometric shift (I520/I640). This self-calibrating system demonstrates high sensitivity, with detection limits of 18.1 pM (R2 = 0.985) for synthetic DNA and 17.2 pM (R2 = 0.996) for mRNA, and a rapid response time (∼10 min). Importantly, the nanoprobe enabled ratiometric imaging of endogenous BRCA1 mRNA in living CD8+ T cells, revealing a significant increase in the I520/I640 ratio during activation, visually confirming BRCA1 upregulation consistent with RNA-seq data. This work provides a robust assay for T-cell studies and highlights red-emissive germanene as a promising platform for ratiometric biosensing.