Capillary electrophoretic enzyme immunoassay with electrochemical detection for cortisol

A novel capillary electrophoretic enzyme immunoassay with electrochemical detection has been developed and used for the determination of cortisol. In this method, after the competitive enzyme immunoreaction, the free enzyme (horseradish peroxidase, HRP)-labeled cortisol (HRP-cortisol) and the bound enzyme-labeled cortisol (HPR-cortisol-anti-cortisol) were separated in the separation capillary and then catalyzed the enzyme substrate [3,3,5,5-tetramethyl-benzidine dihydrochloride, TMB(Red)] in the reaction capillary. The product of the enzymatic catalysis reaction [TMB(Ox)] was amperometrically detected on a carbon fiber microdisk bundle electrode. A concentration limit of detection (LOD) of 1.7 x 10(-1) mol/l, which corresponds to a mass LOD of 7.8 amol, was achieved with the relative standard deviation of 3.3%. The method has been verified using the cortisol controls.     

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.     

Labelling indole-3-acetic acid with tritium

A simple method of obtaining indole-3-acetic acid/ growth stimulating plant hormone/ labelled with tritium has been described. The preparate has specific radioactivity of 455 MBq/mmol, and 50% yield as compared to the initial amount of used substance.     

Microdetermination of indole-3-acetic acid in vegetable extracts by high-performance liquid chromatography with fluorescence detection

A HPLC assay method for the quantitation of indole-3-acetic acid in vegetable extracts is described, using fluorescence detection. The method, which is based on extraction in the presence of poly(vinylpyrrolidone), preliminary cleanup of the extract using Sep Pak C18, and separation on a silica column, has been optimized for the determination of IAA in grape berries. The procedure described is determined to be accurate, sensitive, and reliable over time. The detection limit for IAA is about 0.1 ng.     

Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction

An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC-MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g⁻¹ fresh weight (IAA) to 0.44 pmol g⁻¹ fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.     

4-Hydroxylindole-acetic acid — The microbiological transformation product of indole-3-acetic acid

Conclusions The isolation of an unknown intermediary in the microbiological transformation of indole-3-acetic acid to 4-hydroxyindole-3-acetic acid may help us to determine the structure of 4-hydroxyindole-3-acetic acid.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 5, pp. 1155–1158, May, 1975.The authors express their gratitude to G. K. Skryabin and M. N. Kolosov for assistance in carrying out the present work.     

Comparative ab initio SCF conformational study of 4-chloro-indole-3-acetic acid and indole-3-acetic acid phytohormones (auxins)

An ab initio conformational analysis of 4-chloroindole-3-acetic acid was performed at the RHF/6-311G * * level. The mirror symmetrical conformer (in which the indole ring is coplanar with the COOH group) is most stable; next in energy are the two conformers with the C—COOH fragment perpendicular to the indole ring with relative energies of 2.72 and 6.69 kJ/mol. H(SINGLEBOND)Cl hydrogen bonding results in only a minor stabilization. The results are combined with those obtained earlier for indole-3-acetic acid and recent biological data regarding auxin (growth hormone) activity. From this, assumptions concerning the biologically active conformation are drawn. © 1996 John Wiley & Sons, Inc.     

Capillary electrophoresis with microwave-enhanced electrochemical detection

A new experimental development concerning microwave-assisted electrochemical detection in conjunction with capillary electrophoresis is presented. Focused microwaves are readily incorporated into end-column detection systems for capillary electrophoresis, they induce strong localised thermal activation at microelectrodes, and they affect and modulate, in particular, signals for chemically irreversible redox processes.     

Water and time dependent interaction of iron(III) with indole-3-acetic acid

The influence of water on the interaction between iron(III) and indole-3-acetic acid (IAA) was studied in different organic solutions using rapid-scan stopped-flow spectrophotometry and rapid-freeze/quench Mössbauer spectroscopy. Measurements were also performed in ethanol–water and acetone–water mixtures. The results showed that the interaction between Fe^III and IAA resulted in dimeric Fe^III–IAA complex within 1 s, followed by a slow second step to give Fe²⁺ and IAA(oxidized). No such products were formed in the absence of water. The visible and Mössbauer spectra reflect the nature of the organic solvent and that of the anion of iron(III) salts.     

Participation of the photosensitizer alpha-terthienyl in the peroxidase-catalyzed oxidation of indole-3-acetic acid

The plant photosensitizer alpha-terthienyl (alpha T) is toxic toward a variety of organisms, and normally requires exposure to ultraviolet-A radiation for activation and singlet molecular oxygen formation. However, some toxicity has also been reported to occur in the dark. One hypothesis that has been proposed to account for this light-independent toxicity is that the sensitizer becomes activated by energy transfer from the excited-state products of enzymatic reactions. We have investigated this hypothesis using the horseradish peroxidase (HRP)-catalyzed oxidation of indole-3-acetic acid (IAA), which generates indole-3-aldehyde in an excited triplet state. Light is emitted during the IAA/HRP reaction at acidic pH, is increased by inclusion of alpha T and is not observed with heat-denatured HRP. The rates of both the oxidation of IAA and the subsidence of light emission are more rapid in the IAA/alpha T/HRP system than with IAA and HRP alone, indicating that the presence of alpha T accelerates the reaction. Bleaching occurs at the wavelength of maximal alpha T absorbance and is promoted by the inclusion of IAA. Readdition of both IAA and alpha T to a spent reaction mixture is required to restore light emission after it has subsided, further suggesting that both are consumed in the reaction. We were unable to detect measurable quantities of singlet molecular oxygen formation in this system. These results do not support the energy transfer hypothesis, but instead are more compatible with a model proposed by Krylov and Chebotareva [Krylov, S. N. and A. B. Chebotareva (1993) FEBS Lett. 324, 6-8] for the co-oxidation of IAA and xanthene dyes.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

A disposable electrochemical sensor for the determination of indole-3-acetic acid based on poly(safranine T)-reduced graphene oxide nanocomposite

A disposable electrochemical sensor for the determination of indole-3-acetic acid (IAA) based on nanocomposites of reduced graphene oxide (rGO) and poly(safranine T) (PST) was reported. The sensor was prepared by coating a rGO film on a pre-anodized graphite electrode (AGE) through dipping-drying and electrodepositing a uniform PST layer on the rGO film. Scanning electron microscopic (SEM) and infrared spectroscopic (IR) characterizations indicated that PST-rGO formed a rough and crumpled composite film on AGE, which exhibited high sensitive response for the oxidation of IAA with 147-fold enhancement of the current signal compared with bare AGE. The voltammetric current has a good linear relationship with IAA concentration in the range 1.0 × 10⁻⁷-7.0 × 10⁻⁶ M, with a low detection limit of 5.0 × 10⁻⁸ M. This sensor has been applied to the determination of IAA in the extract samples of several plant leaves and the recoveries varied in the range of 97.71-103.43%.     

Determination of indole-3-acetic acid and indole-3-butyric acid in mung bean sprouts using high performance liquid chromatography with immobilized Ru(bpy)3-KMnO4 chemiluminescence detection

A novel method for determination of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in an extract from mung bean sprouts using high performance liquid chromatography (HPLC) with chemiluminescence (CL) detection is described. The method is based on the CL reaction of auxin (indole-3-acetic acid and indole-3-butyric acid) with acidic potassium permanganate (KMnO₄) and tris(2,2′-bipyridyl)ruthenium(II), which was immobilized on the cationic ion-exchange resin. The chromatographic separation was performed on a Nucleosil RP-C18 column (i.d.: 250 mm × 4.6 mm, particle size: 5 μm, pore size: 100) with an isocratic mobile phase consisting of methanol-water-acetic acid (45:55:1, v/v/v). At a flow rate of 1.0 mL min⁻¹, the total run time was 20 min. Under the optimal conditions, the linear ranges were 5.0 × 10⁻⁸ to 5.0 × 10⁻⁶ g mL⁻¹ and 5.0 × 10⁻⁷ to 1.0 × 10⁻⁵ g mL⁻¹ for IAA and IBA, respectively. The detection limits were 2.0 × 10⁻⁸ g mL⁻¹ and 2.0 × 10⁻⁷ g mL⁻¹ for IAA and IBA, respectively. The relative standard deviation (RSD) of intra-day were 3.1% and 2.3% (n = 11) for 2 × 10⁻⁶ g mL⁻¹ IAA and 2 × 10⁻⁶ g mL⁻¹ IBA; The relative standard deviations of inter-day precision were 6.9% and 4.9% for 2 × 10⁻⁶ g mL⁻¹ IAA and 2 × 10⁻⁶ g mL⁻¹ IBA. The proposed method had been successfully applied to the determination of auxin in mung bean sprouts.     

Binding behavior of amino acid conjugates of indole-3-acetic acid to immobilized human serum albumin

The affinity of indole-3-acetic acid (IAA), indole-3-propionic acid, indole-3-butyric acid and 24 of their amino acid conjugates to immobilized human serum albumin, as expressed by the retention factor k (determined by HPLC), was dependent on (1) lipophilicity, (2) chirality and (3) functional groups in the amino acid moiety; in some cases conformation plays an additional role. Two lipophilicity-related parameters afforded quantitative correlations with k: retention on a C18 reversed-phase column (experimental approach) and the distance between the hydrophilic and hydrophobic poles of the molecules (in silico approach). Most compounds examined are possible metabolic precursors of IAA, an experimental tumor therapeutic.     

One-step co-electrodeposition of graphene oxide doped poly(hydroxymethylated-3,4-ethylenedioxythiophene) film and its electrochemical studies of indole-3-acetic acid

A novel graphene oxide(GO) doped poly(hydroxymethylated-3,4-ethylenedioxythiophene)(PEDOTM)film has been achieved via one-step co-electrodeposition and utilized for electrochemical studies of indole-3-acetic acid(IAA).The incorporation of GO into PEDOTM film facilitated the electrocatalytic activity and exhibited a favorable interaction between the PEDOTM/GO film and the phytohormone during the oxidation of IAA.Under optimized conditions,differential pulse voltammetry and square wave voltammetry were used for the quantitative analysis of IAA,respectively,each exhibiting a wide linearity range from 0.6 μmol L~(-1) to 10 μmol L~(-1) and 0.05 μmol L~(-1) to 40 μmol L~(-1),good sensitivity with a low detection limit of 0.087 μmol L~(-1) and 0.033 μmol L~(-1) respectively,as well as good stability.With the notable advantages of a green,sensitive method,expeditious response and facile operation,the as-prepared PEDOTM/GO organic-inorganic composite film provides a promising platform for electrochemical studies of IAA.     

Sensitive determination of deuterated and non-deuterated indole-3-acetic acid and 5-hydroxyindole-3-acetic acid by combined capillary gas chromatography-negative-ion chemical ionization mass spectrometry

Sensitive methods for the determination of deuterated and non-deuterated indole-3-acetic acid and 5-hydroxyindole-3-acetic acid by combined capillary gas chromatography-negative-ion chemical ionization mass spectrometry were developed. Indole-3-acetic and 5-hydroxyindole-3-acetic acids were converted into pentafluorobenzyl and trifluoroacetylmethyl derivatives, respectively, after pre-purification by high-performance liquid chromatography. These derivatives were separated by gas chromatography and determined by selected ion monitoring. In the determinations, indole-3-acetic-2,2,2',4',5',6',7'-d7 acid and 5-hydroxyindole-3-acetic-3,3-d2 acid were used as internal standards. The methods developed in this work were used for the determination of deuterated and non-deuterated indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human urine samples collected before and after administration of L-tryptophan-3,3-d2.     

Poly(indole-5-carboxylic acid)-functionalized ZnO nanocomposite for electrochemical DNA hybridization detection

Journal of Solid State Electrochemistry - In this paper, a novel direct DNA electrochemical biosensor was developed for detection of breakpoint cluster region gene and the cellular abl (BCR/ABL)...