Microfluidic Chip-Based Wearable Colorimetric Sensor for Simple and Facile Detection of Sweat Glucose.

This study reports a microfluidic chip-based wearable colorimetric sensor for detecting sweat glucose. The device consisted of five microfluidic channels branching out from the center and connected to the detection microchambers. The microchannels could route the sweat excreted from the epidermis to the microchambers, and each of them was integrated with a check valve to avoid the risk of the backflow of the chemical reagents from the microchamber. The microchambers contained the pre-embedded glucose oxidase (GOD)-peroxidase-o-dianisidine reagents for sensing the glucose in sweat. It was found that the color change caused by the enzymatic oxidation of o-dianisidine could show a more sensitive response to the glucose than that of the conventional GOD-peroxidase-KI system. This sensor could perform five parallel detections at one time. The obtained linear range for sweat glucose was 0.1-0.5 mM with a limit of detection of 0.03 mM. The sensor was also used to detect the glucose in sweat samples from a group of subjects engaged in both fasting and postprandial trials. The results showed that our wearable colorimetric sensor can reveal the subtle differences existing in the sweat glucose concentration after the fasting and the oral glucose uptake.

Caeruloplasmin oxidase activity: measurement in serum by use of o-dianisidine dihydrochloride on a microplate reader.

Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a much lower detection limit compared with immunoassay-based methods. It has found its application in both the diagnosis of Wilson's disease and also in the monitoring of patients' response to treatment. Methods The method previously described in literature was adapted for use on a 96-well plate. Caeruloplasmin oxidase activity results were derived from the equation: caeruloplasmin oxidase activity = (A15-A5) × 185 U/L. Results Repeatability (intra-batch) imprecision ranged from 6 to 15% and intermediate (inter-batch) imprecision varied from 7 to 16% for caeruloplasmin oxidative activities of 14, 29, 45 and 99 U/L. Between 3 and 92 U/L, the assay appeared linear with a regression coefficient R2 = 0.9958. The lower limit of quantification was 4 U/L. Samples were stable over a five-week period at 4℃ and for at least four freeze-thaw cycles. There was a statistically significant difference between the areas under ROC curve for copper-to-caeruloplasmin ratios between caeruloplasmin oxidative activity and immunoassay-based methods ( P < 0.0171). The reference interval for caeruloplasmin activity was determined to be 12-166 U/L. Conclusions Using the oxidative assay provides a cost-effective means of estimating caeruloplasmin concentrations. The method is easily adaptable to a 96-well plate format that facilitates high throughput of samples in a busy laboratory. The enzymatic method is more sensitive and specific for differentiating between Wilson's and non-Wilson's when compared with immunoassay-based methods.

Spectrophotometric determination of gold(III) in forensic and pharmaceutical samples and results complemented with ICP AES and EDXRF analysis.

Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the system 1 it oxidizes o-dianisidine, in the system 2 it oxidizes catechol followed by its coupling with o-dianisidine, in the system 3 it oxidizes catechol followed by its coupling with aniline sulphate forming dye products with respective λmax 446nm, 540nm, and 505nm. All the three systems were optimized and analytical parameters were calculated. The molar absorptivity values were 9.27×104, 1.97×104 and 1.62×104 respectively for the systems 1, 2 and 3 with the corresponding Sandell sensitivity values (µgcm-2), 0.0021, 0.0096 and 0.011. The optimized systems were used for the determination of gold present in some forensic jewellery and pharmaceutical samples and the results obtained were compared with the results of all samples determined by Inductively Coupled Plasma - Atomic Emission Spectrometric method and a few of them were also complemented by Energy Dispersive X-Ray Fluorescent spectral analysis.

Fabrication, characterization and application of laccase-nylon 6,6/Fe3+ composite nanofibrous membrane for 3,3'-dimethoxybenzidine detoxification.

In this study, laccase was immobilized on nylon 6,6/Fe3+ composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe3+ particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L-1 within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.

A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation.

Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.

Measurement of plasma hemoglobin peroxidase activity.

We described a spectrophotometric method for measuring hemoglobin peroxidase activity in human plasma using o-dianisidine (o-DA) as the substrate and myeloperoxidase specific inhibitor 4-aminobensoic acid hydrazide (ruling out the probable contribution of myeloperoxidase to the measured parameter value). The optimal conditions (pH 5.5; 2 mM H2O2) have been determined, at which hemoglobin makes the main contribution to plasma oxidation of o-DA. A significant positive correlation between hemoglobin peroxidase activity measured by the spectrophotometric method and hemoglobin level measured by the pyridine hemochromogenic method has been detected (r=0.624; p<0.01) in plasma specimens from 16 donors. Plasma hemoglobin peroxidase activities were measured in healthy individuals and patients with type 2 diabetes mellitus and coronary heart disease. High plasma hemoglobin peroxidase activities in both groups of patients indicates disorders in the mechanisms of clearance of hemoglobin and its highly reactive derivatives and can serve as specific markers of diseases associated with oxidative stress.

Amplified detection of microRNA based on ruthenium oxide nanoparticle-initiated deposition of an insulating film.

A highly sensitive microRNA (miRNA) biosensor that employs ruthenium oxide nanoparticle (RuO(2) NP)-initiated polymerization of 3,3'-dimethoxybenzidine (DB) and miRNA-templated deposition of an insulating poly(3,3'-dimethoxybenzidine) (PDB) film is described in this work. The biosensor was made of a mixed monolayer of oligonucleotide capture probes (CPs) and 4-mercaptoaniline on a gold electrode. Following hybridization with a RuO(2) NP-tagged target miRNA, a mixture of DB/H(2)O(2) in pH 5.0 0.10 M acetate buffer was applied to the biosensor. The RuO(2) NPs serve as polymerization initiator/catalyst for the polymerization of DB. And the hybridized anionic miRNA strands and free CPs serve as templates, guiding the deposition of PDB. The amount of the deposited PDB and its insulating power directly correlated to the concentration of the target miRNA in solution. Electrochemical impedance spectroscopic tests showed that a linear charge-transfer resistance-concentration relationship from 6.0 fM to 2.0 pM was attained after 60 min of incubation in the DB/H(2)O(2) mixture. There was no cross-hybridization between pre-miRNA and mature miRNA and very little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. This impedance-based biosensor offers an attractive alternative for miRNA expression profiling and may enable the development of a portable multiplexing miRNA profiling system.

[New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma].

A novel method for spectrometrical measurement of myeloperoxidase (MPO) activity in plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, including the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or (4-aminobenzoyl)hydrazide, are added to measure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2-250 ng/ml. A direct and significant (P < 0.0001) correlation was observed between the MPO activities measured spectrometrically and by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measurement in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mechanisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases.

Development of new methodologies for on-line determination of the bromate ion in samples of water subjected to ozonation treatment.

The bromate ion has been identified as an inorganic disinfection by-product (DPB) in water subjected to purification treatment. Its presence arises from the application of oxidation processes to water containing bromide through the action of oxidant agents such as the ozone used in ozonation processes. This ion has been identified as a possible carcinogen by the U.S. Environmental Protection Agency, which recommends a maximum concentration of 10 microgL(-1). The literature reports a broad range of methods for the analysis of bromate in water, among which ion chromatography is the one most widely used, with different detection systems. However, most of the methods described to date require state-of-the-art technology and costly instrumentation, such that they are not readily adaptable to routine analyses. The present work reports a procedure for the spectrophotometric determination of bromate based on the bromination reaction of 3-3' dimethoxybenzidine, o-dianisidine (ODA). The reaction is based on the formation of Br2 in the presence of excess bromide and the later bromination of ODA, generating a product that absorbs at 450 nm. The procedure was set up with Flow Injection Analysis and allows the determination of the analyte in the 8 microg L(-1)-3.3 mg L(-1) range, with a detection limit of 6.0 microg L(-1) and relative standard deviations (n=12, [BrO3-]=8.0 and 30.0 microg L(-1)) of 4.8% and 2.9%, respectively. The determination rate was 10-11 samples/hour.

Effect of zinc ion on peroxidase activity of serum in cow.

In this study, for clarifying some possible mechanism of zinc toxicity, the effect of increasing amounts of Zn2+ ion on peroxidase activity was investigated in vitro in serum of cow. The H2O2-mediated oxidation of o-dianisidine was used to assess the peroxidase activity. Results show that after preincubation of serum with 0.2-20 mM Zn2+ concentration for 5 min, peroxidase activity was inhibited compared to the control and decreased rapidly with increasing metal concentrations. The enzyme was completely inhibited after 5 min preincubation in 30 mM Zn2+. When the preincubation of serum and Zn2+ was prolonged to 30 and 60 min, the enzymatic activity decreased more rapidly with increasing metal concentration. Extended exposure of the enzyme to lower concentrations of the metal brought about the same effect as shorter exposure to higher metal concentrations.

Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species.

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3-dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3,5,5-tetra-methylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N,N-diacetylbenzidine were not mutagenic. In addition, 3,3-dichlorobenzidine and 4,4-dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4-Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS.

[Mechanisms of peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate].

Peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1-100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.

Characterization of peroxidase in buckwheat seed.

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.

Hollow gold nanoparticles encapsulating horseradish peroxidase.

Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from Au(shell)AgCl(core) nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows Michaelis-Menten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles.

Enzymes in the cavity of hollow silica nanoparticles.

Due to limitations of the existing preparative methods of hollow nanoparticles by either heating at high temperature (>600 degrees C) or by using strong acid, alkali, or an organic solvent, it was not possible up till now to encapsulate any sensitive organic molecule like enzyme or others inside the cavity of hollow nanoparticles. We have demonstrated a much softer method of preparing hollow silica nanoparticles with horseradish peroxidase (HRP) inside the cavity by synthesizing HRP-doped core-shell silica-coated silver chloride nanoparticles and finally leaching out silver chloride with dilute ammonia at low temperatures. TEM pictures showed the hollow cavity inside the nanoparticles. The enzyme entrapped in these particles was active. The turnover number of HRP entrapped into these hollow particles and dispersed in aqueous buffer (pH 7.2) (k(cat) = 2.56 x 10(6) s(-1)) was found to be less than that of free enzyme in aqueous buffer (k(cat) = 6.133 x 10(7) s(-1)) but higher than that of HRP entrapped in solid-core silica nanoparticles and dispersed in aqueous buffer (k(cat) = 1.05 x 10(5) s(-1)). The result showed that hollow nanoparticles could be prepared using soft chemical methods and sensitive chemicals like active enzyme could be entrapped in the cavities and it retains its activity.

Oxidation of benzidine and its derivatives by thyroid peroxidase.

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.

A novel automated method to measure total antioxidant response against potent free radical reactions.

OBJECTIVES: Oxidative damage of biomolecules occurs as a result of potent free radical reactions. In this study, a novel, colorimetric and fully automated method for measuring total antioxidant response (TAR) against potent free radical reactions is described. DESIGN AND METHODS: Potent free radical reactions were initiated with the production of hydroxyl radical (OH(*)) via Fenton reaction, and the rate of the reactions was monitored by following the absorbance of colored dianisidyl radicals. Ortho-dianisidine (10 mM) and ferrous ammonium sulfate (45 microM) were dissolved in KCl/HCl solution (75 mM, pH 1.8). This mixture was named as Reagent 1 and hydrogen peroxide solution (7.5 mM) as Reagent 2. The OH(*), produced by mixing of R1 and R2, oxidized o-dianisidine molecules into dianisidyl radicals, leading to a bright yellow-brown color development within seconds. Antioxidants, present in the sample, suppressed the color formation to a degree that is proportional to their concentrations. The method was applied to an automated analyzer and analytical performance characteristics of the assay were determined. RESULTS: Vitamin C and Trolox, reduced glutathione, bilirubin, uric acid and (+/-)-catechin solutions suppressed the color formation depending on their concentrations. Serum TAR against potent free radical reactions was lower in patients with chronic renal failure (1.13 +/- 0.21 mmol Trolox equiv./l) and was higher in the individuals with neonatal icterus (2.82 +/- 1.18 mmol Trolox equiv./l) than in healthy subjects (1.54 +/- 0.15 mmol Trolox equiv./l). CONCLUSIONS: The easy, inexpensive and fully automated method described can be used to measure TAR of samples against potent free radical reactions.

[Effect of temperature on structure and functional properties of horseradish peroxidase]. / Struktura i funktsional'nye svoistva peroksidazy khrena pri temperaturnom vozdeistvii.

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.