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  • br Acknowledgements This work was financially supported

    2021-07-16


    Acknowledgements This work was financially supported by Industrial Technology Institute, Sri Lanka through funds received from Sri Lankan Government Treasury (TG13/69). G. D. Liyanaarachchi received postgraduate research scholarship grant from National Science Foundation Sri Lanka (NSF/SCH/2017/06).
    Introduction Human neutrophil elastase (HNE), a serine protease secreted by polymorphonuclear neutrophils (PMNs), plays an important role in many physiological and pathological processes (Korkmaz et al., 2008). The proteolytic activity of this enzyme contributes to the body's defence against infectious agents by promoting the destruction of pathogenic bacteria (Meyer-Hoffert and Wiedow, 2011, Nathan, 2006). High levels of unregulated HNE have also been associated with the inflammatory state found in a wide range of acute and chronic diseases (Kolaczkowska and Kubes, 2013, Pham, 2006) with excess HNE causing extracellular matrix degradation, cellular receptor cleavage and healthy tissue disruption (Abe et al., 2009, Chua and Laurent, 2006, Shapiro, 2002). Furthermore, recent studies have suggested a direct role for HNE in promoting tumour proliferation and Histamine Phosphate receptor (Galdiero et al., 2013, Sato et al., 2006). HNE is therefore a potential diagnostic marker for a number of disease states and detecting it with high sensitivity is clinically important (Henriksen and Sallenave, 2008, Ho et al., 2014, Korkmaz et al., 2010). Common methods to monitor HNE levels rely on immunoassays (de la Rebière de Pouyade et al., 2010, Dunn et al., 1985) and aptamer or peptide-based sensors typically labelled with fluorescent reporters (Avlonitis et al., 2013, Bai et al., 2017, Ferreira et al., 2017, He et al., 2010). In this context, it is interesting to note that electrochemical peptide-based biosensors have proven to be valuable tools for the detection of protease activity (Anne et al., 2012, Liu et al., 2006, Shin et al., 2013, Swisher et al., 2015, Swisher et al., 2014, Swisher et al., 2013) – as well as a wider range of applications (Huang et al., 2016, Li et al., 2015, Li et al., 2014, Puiu et al., 2014). Recently, our research group reported an electrochemical peptide-based biosensor which used self-assembled monolayers (SAMs) on gold electrodes for the detection of the model protease trypsin (González-Fernández et al., 2016). This constituted a short peptide sequence, which acts as substrate for the target enzyme, methylene blue as redox reporter and a polyethylene glycol (PEG) spacer that was shown to be important in tuning both the anti-fouling properties and the probe's flexibility (González-Fernández et al., 2018). Upon enzymatic cleavage, a redox-labelled probe fragment is released, leading to a measurable decrease in the electrochemical signal. This system provided a sensitive platform with a clinically relevant limit of detection (LOD) of 250 pM. Building on this approach, here we target the translation of this model system to the detection of HNE in biological (human blood) samples through the development of a novel methylene blue-tagged peptide-based biosensor with an HNE-specific cleavage sequence employing ternary SAMs on a gold surface. This is the first reported example of HNE detection based on a reagent-free labelled electrochemical strategy.
    Materials and methods
    Results and discussion The sensing platform was based on the immobilisation of a redox-labelled peptide sequence, which contained a specific cleavage site for the enzyme, on a gold surface as a SAM. The peptide sequence (APEEIMRRQ) has been reported as a highly specific HNE cleavable sequence in a fluorescent assay for HNE detection (Avlonitis et al., 2013). A novel peptide probe has been designed and synthesised, adding further functionality to this previously reported sequence. In order to generate an HNE electrochemical probe, a methylene blue redox tag was attached to the amino-terminus of the peptide with the addition of a 2-unit ethylene glycol moiety (PEG-2) and a cysteine at the carboxy-terminus, which enables facile SAM immobilisation of the probe through the formation of a S-Au bond (Fig. 1A). A probe containing the equivalent non-cleavable D-amino acid sequence was also synthesised as a negative control probe. The sensing mechanism relies on specific enzyme-catalysed cleavage followed by the release of the labelled peptide fragment from the SAM-modified gold surface into solution (Fig. 1B). This results in a decrease of the electrochemical signal, which is interrogated before and during enzyme exposure by square wave voltammetry (SWV) and presented as a negative % signal change Histamine Phosphate receptor (a decrease in the relative peak SWV current with respect to the initial value recorded before enzyme addition). The cleavage site, immediately after the methionine residue, was confirmed in solution through cleavage fragment mass analyses by MALDI-TOF MS (Fig. S1). The sensing surfaces were generated using a previously optimised ternary-SAM (T-SAM) configuration (González-Fernández et al., 2016) established as showing enhanced SAM biosensing, according to the protocol detailed in Section 2.3.2. This is denoted a ternary-SAM as it is composed of 3 different thiols on the gold surface: the peptide-probe, a co-adsorbed pegylated dithiol and mercaptohexanol.