A simple and highly repeatable colorimetric toxicity assay method using 2,6-dichlorophenolindophenol as the redox color indicator and whole eukaryote cells

Abstract

A simple and highly reproducible toxicity assay method was studied by employing 2,6-dichlorophenolindophenol (DCIP) as a redox color indicator, baker's yeast Saccharomyces cerevisiae, and a thermostable three-consecutive-stir unit. The absorbance of DCIP was decreased by increasing the metabolism activity of S. cerevisiae to intake glucose as an organic substance. By optimizing the measurement conditions, we obtained highly sensitive responses to glucose between 0.75 and 30 mg/L (eight points, n = 3) with an incubation time of the reaction mixture of 10 min at 30 degrees C. An excellent value of 1.15% was obtained as the average of the repeatability from eight points. Next, for the characterization of this method, we investigated the influence on the colorimetric response of dissolved substances, such as inorganic ions and surfactants, in natural water. Furthermore, the colorimetric responses to several toxicants were examined using Cu2+, Mn2+, Zn2+, Cr3+, and Fe3+ as heavy-metal ions and simazine as an agricultural chemical. As a result, notable colorimetric responses were obtained for Cu2+ and Mn2+ at several concentrations, and the results were compared with those obtained using river water as a real sample. In the stability test, responses to 30 mg/L glucose were obtained for 28 days when the yeast cell suspension was stored at 4 degrees C (response reduction, 43.9%; average of the relative standard deviation for nine testing days, 22.7%; average of repeatability, 1.01%).

The principles and applications of nano-diagnosis system for a nano-biosensor

“…All of them could be composed as sensors by combining various electrodes and transducers that were already described in above. Various sensors, such as receptor sensors using receptors [15], immune sensors using antigen/antibody reactions [16][17][18], cell sensors using animal and plant cells [19][20][21] and tissue sensors using animal and plant tissues [22], have been already developed based on these principles. Since SELEX (systemic evolution of ligands by exponential enrichment), a technology for identifying aptamers, was first introduced by Larry Gold's team in 1990, aptamer-based biosensors have been actively conducted [23,24].…”

A direct determination method for ethanol concentrations in alcoholic beverages employing a eukaryote double-mediator system

“…In recent years, we have actively developed many types of microbial biosensors for use in such environmental fields as soil diagnosis (Hashimoto, Nakamura, Asaga, & Karube, 2008), biological oxygen demand (BOD) measurements (Nakamura et al, , 2007c(Nakamura et al, , 2008a(Nakamura et al, , 2008bYoshida, McNiven, Morita, Nakamura, & Karube, 2002;Yoshida et al, 2000;Yoshida et al, 2001aYoshida et al, , 2001b, toxicity measurements (Ikebukuro, Nakamura, & Karube, 2000, chap. 19; Nakamura et al, 2007d), and composting monitoring (Yoshida et al, 2001c). Recently, microbial biosensors for use in food analyses have been developed.…”

A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

“…cerevisiae [5]. Alternatively the activity of electron transfer reactions can be evaluated by artificial electron acceptors, and tetrazolium salts [8,9,18,19], resazurin (AlamarBlue) [7,20] and various quinoid compounds [6,21,22,23] have been used to detect the viability of microbial and mammalian cells via color changes, which are usually related to the reduction of these compounds. However, also electrochemical detection methods are applicable, when the reduced electron acceptor can be oxidized at electrodes.…”

“…2,6-dichlorophenolindophenol (DCIP) is one of the compounds allowing both spectrophotometric [22,23] and voltammetric [24] detection of cellular reactions. It is known as photometric pH and redox indicator [25].…”