RESUMEN
A unique ratiometric MALDI-MS strategy is proposed for the convenient and reliable quantitation of alkaline phosphatase based on the homogeneous enzymatic cleavage of a coded phosphopeptide (CPP)-triggered double-signal output. The dynamic range can be tuned by simply adjusting the primary concentration of CPP. The proposed strategy is also capable of being challenged by real human serum, and thus it may offer a wonderful approach for the convenient identification and quantitation of various enzyme activities in clinical diagnosis.
Asunto(s)
Fosfatasa Alcalina/sangre , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/química , Ácido Edético/química , Inhibidores Enzimáticos/química , Humanos , Límite de Detección , Fosfopéptidos/análisis , Fosfopéptidos/química , Prueba de Estudio Conceptual , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A novel ratiometric electrochemical biosensing strategy based on T7 exonuclease (T7 Exo)-assisted homogenous target recycling coupling hairpin assembly triggered dual-signal output was proposed for the accurate and sensitive detection of microRNA-141 (miRNA-141). Concretely, in the presence of target miRNA, abundant signal transduction probes were released via the T7 Exo-assisted homogenous target recycling amplification, which could be captured by the specially designed ferrocene-labeled hairpin probe (Fc-H1) on -electrode interface and triggered the nonenzymatic catalytic hairpin assembly (Fc-H1 + MB-H2) to realize the cascade signal amplification and dual-signal output. Through such a conformational change process, the electrochemical signal of Fc (IFc) and MB (IMB) is proportionally and substantially decreased and increased. Therefore, the signal ratio of IMB/IFc can be employed to accurately reflect the true level of original miRNA. Benefiting from the efficient integration of the T7 Exo-assisted target recycle, nonenzymatic hairpin assembly and dual-signal output mode, the proposed sensor could realize the amplified detection of miRNA-141 effectively with a wide detection range from 1 fM to 100 pM, and a detection limit of 200 aM. Furthermore, it exhibits outstanding sequence specificity to discriminate mismatched RNA, acceptable reproducibility and feasibility for real sample. This strategy effectively integrated the advantages of multiple amplification and ratiometric output modes, which could provide an accurate and efficient method in biosensing and clinical diagnosis.
