Washing-Free Displacement Immunosensor for Cortisol in Human Serum Containing Numerous Interfering Species

Abstract

Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)Cl between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN), and Ru(NH), the electrochemical and redox cycling behaviors of Os(bpy)Cl are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.

Surface‐Plasmonic‐Field‐Induced Photoredox Catalysis and Mediated Electron Transfer for Washing‐Free DNA Detection

“…In Figure a, the rate of the mediated electron transfer of Ru(NH 3 ) 6 3+ depends on the distance between EY 2− and the Au electrode, which can be used for differentiating the biospecifically bound EY 2− labels from the unbound EY 2− labels . This differentiation is not highly efficient because all the EY 2− labels are excited by light and contribute to the electrochemical signal.…”

“…Distance‐dependent signals have played crucial roles in washing‐free homogeneous and heterogeneous bioassays . When a target (bio)molecule is bound to a biosensing probe or surface, the distance change between a signaling species (or a label that generates a signaling species) and a signal‐enhancing or ‐interfering species (or a signal‐measuring surface) induces significant signal variation.…”

“…The optical near field (i.e., evanescent field) generated under the condition of total internal reflection or surface plasmon resonance can only excite fluorescent labels near a solid surface and cannot excite the labels far from the surface. In washing‐free electrochemical bioassays, distance‐dependent electron transfer, such as i) distance‐dependent direct electron transfer between a signaling label and an electrode and ii) distance‐dependent mediated electron transfer of an electron mediator between a catalytic label and an electrode, has been used . However, the combined use of distance‐dependent electromagnetic radiation and electron transfer has not been reported in washing‐free bioassays.…”

Trypsin Detection Using Electrochemical Reduction‐based Redox Cycling

“…In electrochemical biosensors, electrochemical oxidation is more commonly employed than electrochemical reduction, because it is more difficult to obtain high signal-tobackground ratios in biosensors using electrochemical reduction. [1][2][3][4] Redox cycling involving a signaling species such as electrochemical-chemical (EC) redox cycling and electrochemical-enzymatic (EN) redox cycling [5][6][7] can be used to obtain significantly increased electrochemical reduction signals. In electrochemical reduction-based EC and EN redox cycling, a signaling species is reduced at the electrode and is then regenerated by an oxidizing agent dissolved in an aqueous electrolyte solution.…”

Specific and Rapid Glucose Detection Using NAD‐dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

“…The electron mediation rates of ruthenium complexes are slow compared to other electron mediators. On the other hand, Os complexes are affected less by interfering species, and their reaction rates with DI is very fast .…”