Rapid and Sensitive Detection of <i>Aspergillus niger</i> Using a Single-Mediator System Combined with Redox Cycling

다국어 초록 (Multilingual Abstract)

<P>Rapid and sensitive mold detection is becoming increasingly important, especially in indoor environments. Common mold detection methods based on double-mediated electron transfer between an electrode and molds are not highly sensitive and reproducible, although they are rapid and simple. Here, we report a sensitive and reproducible detection method specific to <I>Aspergillus niger</I> (<I>A. niger</I>), based on a single-mediator system combined with electrochemical-chemical (EC) redox cycling. Intracellular NAD(P)H-oxidizing enzymes in molds can convert electro-inactive hydroxy-nitro(so)arenes into electro-active hydroxy-aminoarenes. Since the membrane and wall of <I>A. niger</I> is well permeable to both a substrate (4-nitro-1-naphthol) and a reduced product (4-amino-1-naphthol) in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of <I>A. niger</I> due to two reactions: (i) enzymatic reduction of the substrate to the reduced product and (ii) electrochemical oxidation of the reduced product to an oxidized product. When a reducing agent (NADH) is present in the solution, the oxidized product is reduced back to the reduced product and then electrochemically reoxidized. This EC redox cycling significantly amplifies the electrochemical signal. Moreover, the background level is low and highly reproducible because the substrate and the reducing agent are electro-inactive at an applied potential of 0.20 V. The calculated detection limit for <I>A. niger</I> in a common double-mediator system consisting of Fe(CN)<SUB>6</SUB><SUP>3-</SUP> and menadione is ∼2 × 10<SUP>4</SUP> colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is ∼2 × 10<SUP>3</SUP> CFU/mL, indicating that the newly developed single-mediator system is more sensitive. Importantly, the detection method requires only an incubation period of 10 min and does not require a washing step, an electrode modification step, or a specific probe.</P>
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