RESUMO
The high expression of Spermidine/spermine N1-acetyltransferase (SSAT-1) is an important indicator in early cancer diagnosis. Here, we developed a nanopore-based methodology with γ-cyclodextrin as an adaptor to detect and quantify acetylamantadine, the specific SSAT-1-catalyzed product from amantadine, to accordingly reflect the activity of SSAT-1. We employ γ-cyclodextrin and report that amantadine cannot cause any secondary signals in γ-cyclodextrin-assisted α-HL nanopore, while its acetylation product, acetylamantadine, does. This allows γ-cyclodextrin to practically detect acetylamantadine in the interference of excessive amantadine, superior to the previously reported ß-cyclodextrin. The quantification of acetylamantadine was not interfered with even a 50-fold amantadine and displayed no interference in artificial urine sample analysis, which indicates the good feasibility of this nanopore-based methodology in painless cancer prediagnosis. In addition, the discrimination mechanism is also explored by 2-D nuclear magnetic resonance (NMR) and nanopore experiments with a series of adamantane derivatives with different hydrophilic and hydrophobic groups. We found that both the hydrophobic region matching effect and hydrophilic interactions play a synergistic effect in forming a host-guest complex to further generate the characteristic signals, which may provide insights for the subsequent design and study of drug-cyclodextrin complexes.
Assuntos
Amantadina , Nanoporos , gama-Ciclodextrinas , gama-Ciclodextrinas/química , Humanos , Amantadina/química , Amantadina/análise , NeoplasiasRESUMO
Protein identification and discrimination at the single-molecule level are big challenges. Solid-state nanopores as a sensitive biosensor have been used for protein analysis, although it is difficult to discriminate proteins with similar structures in the traditional discrimination method based on the current blockage fraction. Here, we select ferritin and apo-ferritin as the model proteins that exhibit identical exterior and different interior structures and verify the practicability of their discrimination with flexibility features by the strategy of gradually decreasing the nanopore size. We show that the larger nanopore (relative to the protein size) has no obvious effect on discriminating two proteins. Then, the comparable-sized nanopore plays a key role in discriminating two proteins based on the dwell time and fraction distribution, and the conformational changes of both proteins are also studied with this nanopore. Finally, in the smaller nanopore, the protein molecules are trapped rather than translocated, where two proteins are obviously discriminated through the current fluctuation caused by the vibration of proteins. This strategy has potential in the discrimination of other important similar proteins.
Assuntos
Técnicas Biossensoriais , Nanoporos , Ferritinas , NanotecnologiaRESUMO
Selective recognition of short oligonucleotides at the single-molecule level is particularly important for early disease detection and treatment. In this work, polydopamine (PDA)-coated nanopores were prepared via self-polymerization as a solid-state nanopore sensing platform for the recognition of oligonucleotide C (PolyC). The PDA coating possesses abundant active sites, such as indole, amino, carboxyl, catechol, and quinone structures, which had interactions with short oligonucleotides to slow down the translocation rate. PDA-coated nanopores selectively interact with PolyC20 by virtue of differences in hydrogen bonding forces, generating a larger blocking current, while polyA and polyT demonstrated very small blockings. At the same time, PDA-coated nanopores can sensitively distinguish PolyC with different lengths, such as 20, 14, and 10 nt. The functionalization of PDA on the solid-state nanopore provides an opportunity for the rational design of the recognition surface for biomolecules.