Colorimetric and electrochemical detection of ligase through ligation reaction-induced streptavidin assembly |
| |
Institution: | 1. Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China;2. Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang 455000, China;3. Sanquan College of Xinxiang Medical University, Xinxiang 453003, China |
| |
Abstract: | We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin (SA)-biotin interaction. Sortase A (SrtA) served as the model analyte and two biotinylated peptides (bio-LPETGG and GGGK-bio) were used as the substrates. SrtA-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGK-bio. The ligation product (bio-LPETGGGK-bio) induced the aggregation and color change of SA-modified gold nanoparticles (AuNPs) through the SA-biotin interactions, which could be assayed by the colorimetric method. Furthermore, we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the (SA-bio-LPETGGGK-bio)n assemblies through the same interactions. The above results were further confirmed by atomic force microscopy and fluorescent imaging. The insulated assemblies were in-situ fabricated at the SA-modified gold electrode, thus hindering the electron transfer of Fe(CN)6]3?/4? and leading to an increase in the electron-transfer resistance. The capability of the method for the detection of SrtA both in vitro and Staphylococcus aureus (S. aureus) has been demonstrated. SrtA with a concentration down to 1 pmol/L has been determined by the electrochemical analysis, which is lower than that achieved by the colorimetric assay (50 pmol/L). By integrating the advantages of homogeneous reaction and heterogeneous detection, the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|