Colorimetric and electrochemical detection of ligase through ligation reaction-induced streptavidin assembly

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(Srt A)served as the model analyte and two biotinylated peptides(bio-LPETGG and GGGK-bio)were used as the substrates.Srt A-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGKbio.The ligation product(bio-LPETGGGK-bio)induced the aggregation and color change of SA-modified gold nanoparticles(Au NPs)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)nassemblies 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 Srt A both in vitro and Staphylococcus aureus(S.aureus)has been demonstrated.Srt A 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.

Cu-Cu low-temperature diffusion bonding by spark plasma sintering:Void closure mechanism and mechanical properties

In this study,combining the single point diamond turning(SPDT)and spark plasma sintering(SPS),we achieved high-strength diffusion bonding of copper at an ultra-low temperature of 202℃(0.35 T_(m),T_(m):absolute temperature of the melting point).Additionally,the closure mechanism of interfacial micro-and nano-voids during the Cu-Cu SPS diffusion bonding is systematically revealed for the first time.For micro-voids,the pulsed current is found to induce additional diffusion flux and plastic deformation,thereby facilitating the void closure.Molecular dynamics(MD)simulation revealed that at the atomic scale,high-energy Cu atoms induced by the pulsed current can significantly promote the diffusion of low-energy atoms in their vicinity and accelerate the void closure.This study also proposes a novel“evaporation-deposition”nano-void closure mechanism for the previously unstudied nano-void closure process.The results show that the synergistic effect of the pulsed current and nanoscale surface rough-ness can significantly improve joint strength.At a low temperature of 405℃(0.5 T_(m)),on combining the computerized numerical control(CNC)turning and SPS diffusion bonding,the joint strength can reach 212 MPa,while that for the joint obtained by traditional hot pressing diffusion bonding at the same tem-perature is only 47 MPa.We obtained an ultra-high joint strength of 271 MPa using the combined process of SPDT and SPS diffusion bonding at an ultra-low temperature of 202℃(0.35 T_(m)),which is approximately 600℃ lower than the traditional diffusion bonding process temperature of 800℃(0.79 T_(m)).To sum up,this study provides a novel method and theoretical support for realizing low-temperature high-strength diffusion bonding.