A novel electrochemical aptamer–antibody sandwich assay for lysozyme detection

Abstract

In this paper, we have reported a novel electrochemical aptamer-antibody based sandwich biosensor for the detection of lysozyme. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (lysozyme), a biotinylated antibody was used to complete the sandwich format. The subsequent additions of avidin-alkaline phosphatase as an enzyme label, and a 1-naphthyl phosphate substrate (1-NPP) allowed us to determine the concentration of lysozyme (Lys) via Differential Pulse Voltammetry (DPV) of the generated enzyme reaction product, 1-naphthol. Using this strategy, a wide detection range from 5 fM to 5 nM was obtained for a target lysozyme, with a detection limit of 4.3 fM. The control experiments were carried out by using bovine serum albumin (BSA), cytochrome c and casein. The results showed that the proposed biosensor had good specificity, stability and reproducibility for lysozyme analysis. In addition, the biosensor was applied for detecting lysozyme in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for lysozyme detection. This implies that the proposed sandwich biosensor is a promising analytical tool for the analysis of lysozyme in real samples.

Aptamer-Based Electrochemical Sensing of Lysozyme

“…Urinary lysozyme levels have become an indicator of damage to renal tubular cells with diagnostic value in monocytic or myelomonocytic leukemia [42,43]. However, only a few reports show the sensitivity and selectivity required for sensing in real biological samples, achieved using different signal amplification strategies [44][45][46][47]. A rich and easily available source of lysozyme is the egg white of birds.…”

“…covalent linkage of the aptamer to carboxylic-acid functions present on the electrode surface ( Figure 2B) [44,63,77] …”

“…Moving forward from the above direct assay, a sandwich between the surface linked 6 ] 3+ detectable by CV (e.g., as illustrated in [87]); (B) formation of the aptamer-lysozyme complex creates a barrier for the electron transfer of (Fe(CN) 6 ] 4´/3´i n solution proportional to lysozyme concentration and detectable by EIS [63]; (C) sandwich assay for lysozyme analysis using amplification with a lysozyme antibody labeled with alkaline phosphatase [44]; (D) competitive assay where free lysozyme in solution displaces quantum dot-tagged lysozyme, previously bound to surface-immobilized aptamer (e.g., as illustrated in [94]). …”

“…For instance, aptasensors for dual detection of lysozyme and interferon gamma [90] or thrombin [89,94] have been developed based on this principle. [63]; (C) sandwich assay for lysozyme analysis using amplification with a lysozyme antibody labeled with alkaline phosphatase [44]; (D) competitive assay where free lysozyme in solution displaces quantum dot-tagged lysozyme, previously bound to surfaceimmobilized aptamer (e.g., as illustrated in [94]). …”

“…For Strategy (2), an appropriate number of carboxylic functional groups can be introduced on the electrode surface by modification with nanocomposites of graphene oxide-chitosan [61,77], or reduction of diazonium salts of p-aminobenzoic acid on carbon electrodes [44,63], or by attaching vertically-aligned nitrogen-doped carbon nanotubes (VA-NCNTs) [73]. Rohrbach et al modified screen-printed graphite electrodes with multi-walled carbon nanotubes (MWCNTs) containing about 5% carboxylic acid groups [82].…”