Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag + to an amine compound, which can directly reduce Ag + . NADH acts as a reducing agent for the indirect reduction of Ag + via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH 2 -N) in the presence of DT-D. NH 2 -N initiates two redox cycling processes: NH 2 -N, along with Ag + and NADH, are involved in the CC redox cycling, whereas NH 2 -N, along with Ag + , DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as ∼100 fg/ mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications.
The commonly required properties of diffusive electron mediators for point-of-care testing are rapid dissolubility, high stability, and moderate formal potential in aqueous solutions. Inspired by nature, various quinone-containing electron mediators have been developed; however, satisfying all these requirements remains a challenge. Herein, a strategic design toward quinones incorporating sulfonated thioether and nitrogen-containing heteroarene moieties as solubilizing, stabilizing, and formal potential-modulating groups is reported. A systematic investigation reveals that di(thioether sulfonate)-substituted quinoline-1,4-dione (QLS) and quinoxaline-1,4-dione (QXS) display water solubilities of ≈1 m and are rapidly dissoluble. By finely balancing the electron-donating effect of the thioethers and the electron-withdrawing effect of the nitrogen atom, formal potentials suitable for electrochemical biosensors are achieved with QLS and QXS (−0.15 and −0.09 V vs Ag/AgCl, respectively, at pH 7.4). QLS is stable for >1 d in PBS (pH 7.4) and for 1 h in tris buffer (pH 9.0), which is sufficient for point-of-care testing. Furthermore, QLS, with its high electron mediation ability, is successfully used in biosensors for sensitive detection of glucose and parathyroid hormone, demonstrating detection limits of ≈0.3 × 10 −3 m and ≈2 pg mL −1 , respectively. This strategy produces organic electron mediators exhibiting rapid dissolution and high stability, and will find broad application beyond quinone-based biosensors.
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