Ultrasensitive Biocoded SERS Immunoassay for Tracing Molecular Gatekeeper Mfn2 Expression within Single Cells during Stem Cell Differentiation

Mitochondrial fusion protein 2 (Mfn2) as a molecular gatekeeper in mitochondria plays a momentous role in regulating mitochondrial morphology and function to further influence cell behavior. However, ultrasensitive and in situ measurement of low-abundance Mfn2 at the single-cell level remains a challenge, especially during external stress. Herein, an ultrasensitive and "turn-on"-type immunoassay platform for analysis of Mfn2 expression at the single-cell level during the dental pulp stem cell (DPSC) differentiation process was exploited through surface-enhanced Raman spectroscopy (SERS) as a readout modality. In this system, the detection sensitivity of Mfn2 is markedly enhanced through the generation of gap-plasmon "hot spots" caused by the biocoded SERS nanoprobes consisting of AuNPs and AgNPs following the affinity binding of Mfn2. The developed SERS immunoassay exhibited a wide linear relationship for Mfn2, ranging from 0.5 ng/mL to 5 μg/mL. Notably, the biocoded plasmonic nanoprobes reacting with Mfn2 can self-assemble to form an SERS immunoassay biosensor within single cells, which enabled sensitive measurement of Mfn2 expressions during the DPSC differentiation process evoked by electric-impulse stimulation (EIS). Mechanistically, the Mfn2 expression within DPSCs showed palpable downregulation during the EIS-induced differentiation process. The work revealed at the single-cell level a previously unknown role of Mfn2 in DPSC differentiation during the EIS process. The developed SERS immunoassay sensing platform is promising for the early diagnosis of diseases associated with Mfn2.