One-stone-four-birds strategy to construct magnetic mesoporous nano-ratio fluorescence sensor for efficient discovery of PSD95-nNOS uncouplers

It is effective through uncoupling postsynaptic density protein-95 (PSD95) and neuronal nitric oxide synthase (nNOS) for treating ischemic stroke with high disability, mortality and recurrence rate. However, there are no reliable uncouplers for PSD95-nNOS in clinical practice, and the established screening models for discovering PSD95-nNOS uncouplers have high costs and interference. In this study, epoxy functional fluorescent magnetic mesoporous silica (EFMMS) was synthesized by combining carbon dots (CDs), magnetic mesoporous silica and epoxy functionalization. Ni-EFMMS was further constructed by loading nickel ions to simultaneously purify and covalently immobilize His-tagged neuronal nitric oxide synthase (His-nNOS) and then couple with green fluorescent proteins (GFP) labeled PSD95, finally fabricating nano-biosensor (PSD95-nNOS/Ni-EFMMS). The nanomaterial was well constructed and characterized, and it distinctly constituted stable ratio fluorescence between CDs and GFP-PSD95. The ratio fluorescence intensity of PSD95-nNOS/Ni-EFMMS remained stable even after separately mixing with metal ions, amino acids and natural impurities, and the loss of ratio fluorescence intensity was less than 20% even at pH 4 or 10. PSD95-nNOS/Ni-EFMMS still kept the ratio fluorescence intensity above 90% after being recycled for seven times. Three compounds (gnetol, baicalein and emodin-8- O -β- D -glucoside) with strong uncoupling efficiency were screened from fourteen natural active ingredients. Furthermore, the theoretical feasibility of uncoupling was verified by molecular docking. In general, this study provides a novel “one-stone-four-birds strategy” to fabricate magnetic mesoporous nano-ratio fluorescence sensor for efficiently and accurately discovering PSD95-nNOS uncoupling active substances.