Disposable DNA biosensor with the carbon nanotubes–polyethyleneimine interface at a screen-printed carbon electrode for tests of DNA layer damage by quinazolines

A screen-printed carbon working electrode within a commercially available screen-printed three-electrode assembly was modified by using a composite of multiwalled carbon nanotubes (MWCNT) dispersed in polyethylenimine (PEI) followed by covering with the calf thymus dsDNA layer. Several electrochemical methods were used to characterize the biosensor and to evaluate damage to the surface-attached DNA: square wave voltammetry of the [Ru(bpy)₃]²⁺ redox indicator and mediator of the guanine moiety oxidation, cyclic voltammetry and electrochemical impedance spectroscopy in the presence of the [Fe(CN)₆]^3−/4− indicator in solution. Due to high electroconductivity and large surface area of MWCNT and positive charge of PEI, the MWCNT–PEI composite is an advantageous platform for the DNA immobilization by the polyelectrolyte complexation and its voltammetric and impedimetric detection. In this respect, the MWCNT–PEI interface exhibited better properties than the MWCNT–chitosan one reported from our laboratory previously. A deep DNA layer damage at incubation of the biosensor in quinazoline solution was found, which depends on the quinazoline concentration and incubation time. Figure Impedance spectra for the modified electrodes. Conditions: 1 mM [Fe(CN)₆]^3–/4– in 0.1 M PBS (pH = 7.0), potential amplitude 10 m V, frequency range 12–1×10₄ Hz. Dedicated to Professor Jan Garaj on the occasion of his 75th birthday     

Capacitive biosensor for detection of endotoxin

A capacitive biosensor for the detection of bacterial endotoxin has been developed. Endotoxin-neutralizing protein derived from American horseshoe crab was immobilized to a self-assembled thiol layer on a biosensor transducer (Au). Upon injection of a sample containing endotoxin, a decrease in the observed capacitive signal was registered. Endotoxin could be determined under optimum conditions with a detection limit of 1.0 × 10⁻¹³ M and linearity ranging from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹⁰ M. Good agreement was achieved when applying endotoxin preparations purified from an Escherichia coli cultivation to the capacitive biosensor system, utilizing the conventional method for quantitative endotoxin determination, the Limulus amebocyte lysate test as a reference. The capacitive biosensor method was statistically tested with the Wilcoxon signed rank test, which proved the system is acceptable for the quantitative analysis of bacterial endotoxin (P < 0.05). Figure The flow-injection capacitive biosensor system and the capacitive properties of the transducer surface, where C_SAM is the capacitance change of the self-assembled thiol monolayer, C_P is the capacitance change of the protein layer, C_a is the capacitance change of the analyte layer and C_Total is the total capacitance change measured at the working electrode/solution interface (modified from Limbut et al., 2006. Biosens Bioelectron 22: 233-240)     

An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres

A new kind of magnetic dextran microsphere (MDMS) with uniform shape and narrow diameter distribution has been prepared from magnetic iron nanoparticles and dextran. Horseradish peroxidase (HRP) was successfully immobilized on the surface of an MDMS-modified glassy-carbon electrode (GCE), and the immobilized HRP displayed excellent electrocatalytic activity in the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ). The effects of experimental variables such as the concentration of HQ, solution pH, and the working potential were investigated for optimum analytical performance. This biosensor had a fast response to H₂O₂ of less than 10 s and an excellent linear relationship was obtained in the concentration range 0.20 μmol L⁻¹–0.68 mmol L⁻¹, with a detection limit of 0.078 μmol L⁻¹ (S/N = 3) under the optimum conditions. The response showed Michaelis–Menten behavior at larger H₂O₂ concentrations, and the apparent Michaelis–Menten constant was estimated to be 1.38 mmol L⁻¹. Moreover, the selectivity, stability, and reproducibility of the biosensor were evaluated, with satisfactory results. Figure Amperometric response of the biosensor to successive additions of H₂O₂ and the plot of amperometric response vs. H₂O₂ concentration     

Amperometric hydrogen peroxide sensor based on a sol-gel-derived ceramic carbon composite electrode with toluidine blue covalently immobilized using 3-aminopropyltrimethoxysilane

A carbon composite amperometric hydrogen peroxide sensor has been developed using a sol-gel technique. Toluidine blue (TB), which acts as the redox mediator, was covalently immobilized via glutaraldehyde crosslinking with an organically modified silane, namely 3-aminopropyltrimethoxysilane (APTMOS). Methyltrimethoxysilane (MTMOS) was used as the additional monomer; this controls the hydrophobicity of the electrode surface, thus limiting the wettability. The immobilization of TB within the sol-gel matrix was confirmed with FTIR studies. The sol-gel mixture containing TB immobilized in APTMOS and MTMOS was mixed with graphite powder in order to prepare the carbon composite electrode. The electrode was characterized using voltammetric techniques and its electrocatalytic activity for the reduction of hydrogen peroxide was also studied. The carbon composite electrode has the advantage of sensing H₂O₂ at a lower potential and with a higher sensitivity, and interferences due to ascorbic acid, uric acid and acetaminophen were greatly minimized. The linear range for the determination of H₂O₂ extends from 5.37 × 10⁻⁶ to 6.15 × 10⁻³ M, with a correlation coefficient of 0.9981. The detection limit was found to be 2.15 × 10⁻⁶ M. The covalent immobilization of TB effectively prevents the leakage of the water-soluble mediator during measurements. The modified electrode, aside from electrocatalyzing the reduction of H₂O₂, exhibits distinct advantages in terms of surface renewal in the event of surface fouling, as well as simple preparation, good chemical and mechanical stability, and good reproducibility. Figure Amperometric hydrogen peroxide sensor based on sol-gel-derived ceramic carbon composite electrode with toluidine blue covalently immobilized using 3-aminopropyltrimethoxysilane Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube–cross-linked chitosan composite

A simple method has been devised for immobilization of acetylcholinesterase (AChE)—covalent bonding to a multiwall carbon nanotube (MWNT)–cross-linked chitosan composite (CMC)—and a sensitive amperometric sensor for rapid detection of acetylthiocholine (ATCl) has been based on this. Fourier-transform infrared spectroscopy proved that the native structure of the immobilized enzyme was preserved on this chemically clean and homogeneous composite film, because of the excellent biocompatibility and non-toxicity of chitosan. Glutaraldehyde was used as cross-linker to covalently bond the AChE, and efficiently prevented leakage of the enzyme from the film. Because of the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl, with a value of 132 μmol L⁻¹, forming thiocholine, which was then oxidized to produce a detectable and rapid response. Under optimum conditions the amperometric current increased linearly with the increasing concentration of ATCl in the range 2.0–400 μmol L⁻¹, with a detection limit of 0.10 μmol L⁻¹. Fabrication reproducibility of the sensor was good and the stability was acceptable. The sensor is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis. Abstract     

Room temperature ionic liquid doped DNA network immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

A novel electrochemical H₂O₂ biosensor was constructed by embedding horseradish peroxide (HRP) in a 1-butyl-3-methylimidazolium tetrafluoroborate doped DNA network casting on a gold electrode. The HRP entrapped in the composite system displayed good electrocatalytic response to the reduction of H₂O₂. The composite system could provide both a biocompatible microenvironment for enzymes to keep their good bioactivity and an effective pathway of electron transfer between the redox center of enzymes, H₂O₂ and the electrode surface. Voltammetric and time-based amperometric techniques were applied to characterize the properties of the biosensor. The effects of pH and potential on the amperometric response to H₂O₂ were studied. The biosensor can achieve 95% of the steady-state current within 2 s response to H₂O₂. The detection limit of the biosensor was 3.5 μM, and linear range was from 0.01 to 7.4 mM. Moreover, the biosensor exhibited good sensitivity and stability. The film can also be readily used as an immobilization matrix to entrap other enzymes to prepare other similar biosensors. Figure Horseradish peroxidase (HRP) embedded in a 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF ₄ ) doped DNA network can be used to fabricate a HRP sensor for the determination of H₂O₂     

Amperometric biosensor for the determination of creatine

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine     

Flow-injection solid surface lanthanide-sensitized luminescence sensor for determination of <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 μL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ _ex = 290 nm and λ _em = 546 nm. The method shows a linear range from 0.2 to 6.0 μg mL⁻¹ with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h⁻¹. A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment. Figure Typical emission bands of Tb(III) in a solid-phase PABA–Tb(III) luminescence spectrum     

Chemiluminescent imaging analysis of interferon alpha in serum samples

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (α-IFN) in human serum samples. A typical “sandwich type” immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H₂O₂), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol–H₂O₂–HRP–PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of α-IFN in the range 1.3–156.0 pg mL⁻¹ (R = 0.9991) and the detection limit was 0.8 pg mL⁻¹ (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of α-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA. Figure Procedures of the proposed method     

Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA–bovine thyroglobulin conjugate and OA–N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 μl min⁻¹; flow rate, 25 μl min⁻¹. An assay action limit of 126 ng g⁻¹ was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g⁻¹, which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC–MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r ² = 0.991). Figure Biacore     

Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine

The application of near-infrared (NIR) dyes (λ _em > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04–0.5 μg mL⁻¹ for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL⁻¹ were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6–104% were achieved. Figure Near-infrared probe for protein determination     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study

Radix Scrophulariae (Xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of Xuanshen. The purpose of this study was to develop an HPLC–UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of Xuanshen extract (760 mg kg⁻¹). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid–liquid extraction. Separation was achieved on a Kromasil C₁₈ column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7–103.5% and 96.9–102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of Xuanshen extract were as follows: C _max 1488.7 ± 205.9 and 556.8 ± 94.2 ng mL⁻¹, T _max 2.09 ± 0.31 and (1.48 ± 0.14 h, AUC_0–24 10336.4 ± 1426.8 and 3653.1 ± 456.4 ng h mL⁻¹, 11276.8 ± 1321.4 and 3704.5 ± 398.8 ng h mL⁻¹, and t _1/2 4.9 ± 1.3 and 2.5 ± 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of Radix Scrophulariae extract in rats. Figure Radix Scrophulariae     

New hexamethine–hemicyanine dyes for the development of integrated optochemical sensors

New far-visible absorbing anilino-cyanine dyes have been synthesised for future application as chromoionophores in integrated waveguide absorbance optodes based on bulk optodes. The effect of the heterocycle, of the substitution of the heterocyclic nitrogen and of the type of heptamethine central ring on the pK _a values (4.3–8.2 in ethanol–water solutions and 9.5–11.0 in plasticised PVC membranes), on the spectroscopic characteristics of the dye and on photostability is discussed. pH-selective bulk optodes have been formulated as a first approach to develop ion-selective optodes, and sensitivity, repeatability, lifetime and response time have been determined. The dyes show good analytical behaviour for use as chromoionophores for the development of ion-selective optodes. Reversible (80–87%), fast (tr_90% = 0.94–2.28 min) and pH-sensitive membranes (slopes of 0.09–0.23 ΔAbs·pHdec^–1, absorbance range 0.19–0.53) have been obtained. Moreover, they exhibit good spectroscopic features for employment with integrated optochemical sensors: absorption maxima of the acidic species in plasticised PVC membranes matched those of 650–670-nm LEDs, high molar absorption coefficients ( L mol^–1 cm^–1 and L mol⁻¹ cm⁻¹) and fluorescence. Absorption spectra of the acidic and basic structures of one of the synthesised chromoionophores at different pKa values. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λ_max = 390 nm) and the mutant Y139F emits greenish light (λ _max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca²⁺ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay. Figure Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.     

The potentiometric behavior of polymer-supported metallophthalocyanines used as anion-selective electrodes

Liquid polymer membrane electrodes based on nickel and manganese phthalocyanines were examined for use as anion-selective electrodes. The electrodes were prepared by incorporating the ionophores into plasticized poly(vinyl chloride) membranes, which were directly coated onto the surfaces of graphite electrodes. The resulting electrodes demonstrate near-Nernstian responses over a wide linear range of perchlorate anion (5 × 10⁻⁷ to 1 × 10⁻¹ M). The electrodes have a fast response time, submicromolar detection limits (5 × 10⁻⁷ M perchlorate), and could be used over a wide pH range of 3.5–10. The influences of lipophilic cationic and anionic additives on the response properties of the electrodes were investigated. The proposed sensors revealed high selectivity for perchlorate over a number of common inorganic and organic anions. The highest selectivity was observed for the electrode based on manganese phthalocyanine in the presence of the lipophilic anionic additive sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate. Application of the electrodes to determine perchlorate in tap water and human urine is also reported.      

Photofragmentation of nitro-based explosives with chemiluminescence detection

A simple, fast, reliable, sensitive and potentially portable explosive detection device was developed employing laser photofragmentation (PF) followed by heterogeneous chemiluminescence (CL) detection. The PF process involves the release of NO_{x(x = 1,2)} moieties from explosive compounds such as TNT, RDX, and PETN through a stepwise excitation–dissociation process using a 193 nm ArF laser. The NO_{x(x = 1,2)} produced upon PF is subsequently detected by its CL reaction with basic luminol solution. The intensity of the CL signal was detected by a thermoelectrically cooled photomultiplier tube with high quantum efficiency and negligible dark current counts. The system was able to detect trace amounts of explosives in various forms in real time under ambient conditions. Detection limits of 3 ppbv for PETN, 2 ppbv for RDX, and 34 ppbv for TNT were obtained. It was also demonstrated that the presence of PETN residue within the range of 61 to 186 ng/cm² can be detected at a given signal-to-background ratio of 10 using a few microjoules of laser energy. The technique also demonstrated its potential for the direct analysis of trace explosive in soil. An LOD range of 0.5–4.3 ppm for PETN was established, which is comparable to currently available techniques. Figure Photofragmentation–chemiluminescence detector     

Competitive binding of cadmium by plant thiols: an electrochemical study assisted by multivariate curve resolution

Multivariate curve resolution with alternating least squares (MCR-ALS) has been applied to voltammetric data obtained from analysis of the competitive binding of cysteine (Cys) and cysteine–glycine (Cys-Gly) by Cd(II) as a first approach towards mixtures of phytochelatins and related compounds in natural media. From different starting points, the possibilities of formation of mixed complexes and/or displacements between ligands are investigated. Analysis of the resulting unitary voltammograms and concentration profiles of the resolved components by MCR-ALS suggests that the strongest ligand (Cys-Gly) is able to displace the weakest (Cys) from its metal complexes, whereas this does not happen in the opposite direction. On the other hand, no evidence of Cd mixed-ligand complexes was found. Figure Differential pulse polarograms measured in the independent titrations of 1 × 10^-5 mol L^-1 Cys, 1 × 10^-5 mol L^-1 Cys-Gly, and a mixture of Cys-Gly (0.5 × 10^-5 mol L^-1) and Cys (1 × 10^-5 mol L^-1) with Cd²⁺, at TRIS-HNO₃ buffer (0.1 mol L^-1 and PH 7.5) in the presence of 0.1 mol L^-1 KNO₃     

Biosensor for total cholesterol estimation using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane self-assembled monolayer

Cholesterol oxidase (ChOx), cholesterol esterase (ChEt), and horseradish peroxidase (HRP) have been co-immobilized covalently on a self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTS) deposited on an indium–tin–oxide (ITO) glass surface. These enzyme-modified (ChOx-ChEt-HRP/AEAPTS/ITO) biosensing electrodes have been used to estimate cholesteryl oleate from 10 to 500 mg dL⁻¹. The sensitivity, K _m value, and shelf-life of these ChEt-ChOx-HRP/AEAPTS/ITO biosensing electrodes have been found to be 124 nA mg⁻¹ dL, 95.098 mg dL⁻¹ (1.46 mmol L⁻¹), and ten weeks, respectively. The ChEt-ChOx-HRP/AEAPTS/ITO bio-electrodes have been used to estimate total cholesterol in serum samples. Figure Covalent immobilization of enzymes onto AEAPTS/ITO surface using EDC/NHS chemistry Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous determination of uric acid and ascorbic acid at a ferrocenium–thioglycollate modified electrode

Self-assembled monolayers (SAMS) of chemisorbed thioglycollate on a gold electrode surface have been used as a base interface for the electrostatic adsorption of ferrocenium ion. Electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were used to evaluate the electrochemical properties of the supramolecular film. The bare gold electrode failed to distinguish the oxidation peaks of ascorbic acid (AA) and uric acid (UA) in phosphate buffer solution (PBS, pH 7.0), while the ferricinium–thioglycollate modified electrode could separate them efficiently. In differiential pulse voltammetric measurements, the prepared gold electrode could separate AA and UA signals, allowing the simultaneous determination of AA and UA. Under optimal conditions and within the linear range of 1.0 × 10⁻⁶ to 5.0 × 10⁻⁴ M, the detection limits of AA and UA achieved were 2.0 × 10⁻⁷ and 1.0 × 10⁻⁷ M, respectively. The applicability of the prepared electrode was demonstrated by measuring AA and UA in human urine without any pretreatment. Figure Fabrication process for the modified electrode     

New concept for a toxicity assay based on multiple indexes from the wave shape of damped metabolic oscillation induced in living yeast cells (part I): characterization of the phenomenon

The damped glycolytic oscillation phenomenon occurring in starved cells of the yeast Saccharomyces cerevisiae (NBRC 0565) was characterization for application to a toxicity bioassay. S. cerevisiae was grown under semi-anaerobic conditions. The transient oscillations were observed photometrically as the time course of the fluorescent intensity of reduced pyridine nucleotide resulting from instantaneous addition of glucose to a cell suspension. In this study, simple and reproducible conditions inducing damped oscillations were obtained by modifying a literature method. For estimation of the wave shapes of the damped oscillations we used six indexes. To investigate the total reproducibility as the averaged relative standard deviation (RSD_av) for the six indexes obtained from the wave shapes, the damped oscillations were induced under the optimum conditions and the RSD_av values were calculated as 14% in a buffer cell suspension (n = 62) and 22% in a water cell suspension (n = 78). Finally, the effects of glucose concentration on the six indexes were examined, and all the indexes changed when the glucose concentration was changed. Excellent correlations were obtained between the index of oscillation-state time and the concentration of glucose in a buffer cell suspension (r = 0.9985, 0.5–250 mmol L⁻¹, 10 points) and in a water cell suspension (r = 0.9989, 2.5 μmol L⁻¹–250 mmol L⁻¹, 12 points), respectively. Figure Characterization of damped glycolytic oscillation, (a) typical shape, and (b) its estimation Electronic supplementary material The online version of this article (doi:)contains supplementary material, which is available to authorized users.