A simple and sensitive label-free electrochemiluminescence (ECL) immunosensor based on the use of luminol functionalized gold nanoparticles (luminol-AuNPs) as antibody carriers and sensing platform is described for detecting the acute myocardial infarction biomarker cTnI. The ECL immunosensor was fabricated by the assembly of luminol-AuNPs conjugated with biotinylated antibodies against cTnI (biotin-anti-cTnI-luminol-AuNPs) with the streptavidin coated AuNPs (SA-AuNPs) modified Au electrode directly by virtue of the biotin-SA system. The fabricated sensing platform exhibited stable and strong ECL intensity and could be used for the recognition of target antigen. In the presence of cTnI, a decrease in the ECL intensity was observed. Direct detection of the ECL signal changes during antigen-antibody immunoreactions can be used for the quantification of cTnI. The ECL response exhibited a quite wide dynamic range from 1000 ng mL(-1) down to 0.1 ng mL(-1). The proposed method has been successfully applied in the detection of cTnI in real plasma samples. This protocol is simple, fast, sensitive, specific, stable and reliable. This work reveals that the luminol-AuNPs are excellent sensing platforms for the fabrication of simple and sensitive immunosensors. Moreover, the proposed strategy may also be extended for the detection of other biomarkers, which is of great application potential in clinical and pharmaceutical analysis.
Molecular dynamics simulations are performed to investigate the dynamic properties of wild-type HIV-1 protease and its two multi-drug-resistant variants (Flap + (L10I/G48V/I54V/V82A) and Act (V82T/I84V)) as well as their binding with APV and DRV inhibitors. The hydrophobic interactions between flap and 80 s (80’s) loop residues (mainly I50-I84’ and I50’-I84) play an important role in maintaining the closed conformation of HIV-1 protease. The double mutation in Act variant weakens the hydrophobic interactions, leading to the transition from closed to semi-open conformation of apo Act. APV or DRV binds with HIV-1 protease via both hydrophobic and hydrogen bonding interactions. The hydrophobic interactions from the inhibitor is aimed to the residues of I50 (I50’), I84 (I84’), and V82 (V82’) which create hydrophobic core clusters to further stabilize the closed conformation of flaps, and the hydrogen bonding interactions are mainly focused with the active site of HIV-1 protease. The combined change in the two kinds of protease-inhibitor interactions is correlated with the observed resistance mutations. The present study sheds light on the microscopic mechanism underlying the mutation effects on the dynamics of HIV-1 protease and the inhibition by APV and DRV, providing useful information to the design of more potent and effective HIV-1 protease inhibitors.
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