Diaphorase-Catalyzed Formation of a Formazan Precipitate and Its Electrodissolution for Sensitive Affinity Biosensors

Abstract

Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (∼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.

Overview on the Development of Electrochemical Immunosensors by the Signal Amplification of Enzyme- or Nanozyme-Based Catalysis Plus Redox Cycling

“…DT-D is a flavin-containing oxidoreductase that can catalyze the reduction of redox mediators (e.g., metal complexes, quinones, and nitro(so) compounds) in the presence of NADH or NADPH [ 154 ]. Because of its unique properties, DT-D has been used in EN redox cycling for electrochemical immunosensors.…”

Enzymatic Precipitation of Highly Electroactive and Ion-Transporting Prussian Blue for a Sensitive Electrochemical Immunosensor

“…These precipitated products alleviate issues related to diffusion away from the electrode for sensitive detection and are advantageous for multiplex detection without interference when employing array electrodes. 5−7 Electroactive molecular precipitates can be achieved via three main approaches (Figure 1): (i) low solubility of the product, 5,6,8 (ii) formation of an insoluble conductive polymer derived from the product, 9,10 and (iii) creation of an insoluble coordination polymer from the product. 11−15 In the first scenario, the substrate must be highly soluble, whereas the product needs to be insoluble.…”

“…Electroactive molecular precipitates can be achieved via three main approaches (Figure ): (i) low solubility of the product, ,, (ii) formation of an insoluble conductive polymer derived from the product, , and (iii) creation of an insoluble coordination polymer from the product. − In the first scenario, the substrate must be highly soluble, whereas the product needs to be insoluble. However, achieving this solubility difference through simple chemical modification of the substrate is challenging.…”