ABSTRACT
In this study, a simple, highly sensitive electrochemical biosensor for myoglobin was developed using a myoglobin-specific binding peptide as a sensing probe. A peptide (Myo-3R7, CPSTLGASC, 838 Da) identified by phage display and that specifically binds to myoglobin was covalently immobilized on a gold electrode functionalized via a dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer (SAM). The peptide immobilization was confirmed with fluorescence microarray scanning and cyclic voltammetry (CV). The electrochemical performance of the biosensor with respect to myoglobin was characterized by CV and differential pulse voltammetry (DPV) using Fe(CN)6(3-)/Fe(CN)6(4-) as a redox probe. We successfully detected myoglobin in a broad working range of 17.8 to 1780 ng mL(-1) with a correlation coefficient (R(2)) of 0.998. The estimated limit of detection (LOD) was fairly low, 9.8 ng mL(-1) in 30 min. The electrochemical biosensor based on a myoglobin-specific binding peptide offers sensitivity, selectivity, and rapidity, making it an attractive tool for the early detection of cardiac infarction.
Subject(s)
Biosensing Techniques/methods , Myocardial Infarction/diagnosis , Myoglobin/metabolism , Oligopeptides/metabolism , Acute Disease , Amino Acid Sequence , Early Diagnosis , Electrochemistry , Ferricyanides/chemistry , Gold/chemistry , Humans , Limit of Detection , Models, Molecular , Myoglobin/chemistry , Oligopeptides/chemistry , Protein Conformation , Substrate Specificity , Succinimides/chemistry , Time FactorsABSTRACT
Even low concentrations of endotoxins can be life-threatening. As such, continuous effort has been directed toward the development of sensitive and specific endotoxin detection systems. In this paper, we report the design and fabrication of a new electrochemical endotoxin sensor based on a human recombinant toll-like receptor 4 (rhTLR4) and myeloid differentiation-2 (MD-2) complex. The rhTLR4/MD-2 complex, which specifically binds to endotoxin, was immobilized on gold electrodes through a self-assembled monolayer (SAM) technique involving the use of dithiobis(succinimidyl undecanoate) (DSU). The surface topography of the electrodes at each fabrication stage was characterized with a nanosurface profiler and atomic force microscope (AFM). The electrochemical signals generated from interactions between the rhTLR4/MD-2 complex and the endotoxin were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A linear relationship between the peak current and endotoxin concentration was obtained in the range of 0.0005 to 5 EU/mL with a correlation coefficient (R(2)) of 0.978. The estimated limit of detection (LOD) was fairly low, 0.0002 EU/mL. The rhTLR4/MD-2 based sensors exhibited no current responses to dipalmitoylphosphatidylcholine (DPPC) bearing two lipid chains, which is structurally similar to endotoxin, indicating the high specificity of the sensors to endotoxin.
