Visualizing indoor ozone exposures via o-dianisidine based colorimetric passive sampler.

In this study, we developed a colorimetric ozone passive sampler (OPS) incorporating o-dianisidine, a redox dye, into a polydimethylsiloxane sheet. The reaction between ozone (O3) and o-dianisidine result in a visible yellowish color change. Unlike previous passive methods that rely on nitrate extraction or the color disappearance of indigotrisulfonate, the OPS offered improved recognition of average O3 exposure. To optimize OPS based on time-weighted average (TWA), we extracted and quantified the amount of reacted o-dianisidine after exposing OPS to O3 by varying concentrations (0-200 ppb) within 8 h. Colorimetric changes of OPS were further analyzed by capturing images, and the effective absorbance of blue scale showed the best fit (EAB, R2 =0.997). OPS validation on visual detection assessed by six parameters: limit of detection, limit of quantification, reproducibility, sampling rate, selectivity to interfering gases, and sensitivity to environmental factors. To enhance visibility, the OPS was assembled with coloration exposure guidelines, and a smartphone app was developed to quantify average O3 exposures. We further conducted field tests that showed the significant disparity between O3 concentrations and personal O3 exposures, which is considered more crucial for assessing health risks. The OPS was optimized to monitor O3 exposure levels and raise awareness among workers and occupants regarding invisible indoor hazards.

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.

Treatment of 3,3'-dimethoxybenzidine in sludge by advance oxidation process: Degradation products and toxicity evaluation.

Studies on the oxidation products of organic pollutants and their toxicity in textile dyeing sludge after the sludge was treated by the advance oxidation processes were limited, since textile dyeing sludge was a complicated mixture. For the first time, simulated sludge was used to study the degradation mechanism of 3,3'-dimethoxybenzidine (DMB) during the combined ultrasound-Mn(VII) treatment. The toxicity of DMB and its products was also evaluated. The results indicated that the compositions and microstructures of polyaluminium chloride (PAC)- and polyferric sulphate (PFS)-based simulated sludge were similar to those of real textile dyeing sludge. The optimum conditions of ultrasound-Mn(VII) treatment were: a KMnO4 dosage of 40 µM, an ultrasound power density of 0.36 W cm-3, and a reaction time of 20 min. 98.24% of DMB and 63.04% of total organic carbon (TOC) in the simulated sludge were removed. Six products, that is, 2-nitroanisole, 3-methoxy-4-nitrophenol, vanillylmandelic acid, vanillyl alcohol, m-anisic acid, and benzoic acid, were identified by GC-MS and LC-MS-MS. It was noted that all of these identified products were also detected in the real textile dyeing sludge after the ultrasound-Mn(VII) treatment. All of them were less toxic than DMB. Moreover, 53.30% and 54.80% of toxicity toward the alga Desmodesmus subspicatus and the bacterium Vibrio fischeri were removed in simulated sludge, respectively. Therefore, simulated sludge was helpful for studying a pollutant's degradation mechanism in the complex sludge mixtures. The results would also provide some useful suggestions for the sludge disposal after the sludge was treated by the advance oxidation processes.

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.

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.

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.

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.

"Ready-to-use" hollow nanofiber membrane-based glucose testing strips.

A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 µL of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 °C as long as four months.

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.

D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.

Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson's disease and in hepatic encephalopathy.

Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.

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.

A label-free microRNA biosensor based on DNAzyme-catalyzed and microRNA-guided formation of a thin insulating polymer film.

Herein we report a label-free microRNA (miRNA) biosensor in which the formation of a thin insulating film is used to amplify the analytical signal. Briefly, the biosensor is made of an oligonucleotide-coated gold electrode. After hybridizing with a target miRNA, free capture probe (CP) strands on the biosensor are removed by a nuclease digestion. A second hybridization with an oligonucleotide-tailed DNAzyme is performed to introduce the DNAzyme to the biosensor. The DNAzyme triggers the polymerization of 3,3'-dimethoxybenzidine (DB) in the presence of H2O2 and the hybridized miRNA-CP duplexes serve as templates to guide the deposition of poly (3,3'-dimethoxybenzidine) (PDB). The formation of the insulating PDB film alters the impedance of the biosensor, rendering it readily distinguishable by electrochemical impedance measurements. The accumulative nature of the PDB deposition drastically improves the detectability of the biosensor. A proof-of-concept study is conducted on the detection of miRNAs in total RNA extracted from cultured cells.

Combining the physical adsorption approach and the covalent attachment method to prepare a bifunctional bioreactor.

Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N(2) adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H(2)O(2) as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme.

Influence of smoking on interleukin-1beta level, oxidant status and antioxidant status in gingival crevicular fluid from chronic periodontitis patients before and after periodontal treatment.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the impact of smoking on the relationship between interleukin-1 (IL-1ß) and oxidation in patients with periodontitis and response to nonsurgical periodontal therapy. MATERIAL AND METHODS: Data were obtained from 30 patients with generalized chronic periodontitis (15 smokers and 15 nonsmokers) and from 10 periodontally healthy controls. IL-1ß level, total oxidant status (TOS) and total antioxidant status (TAS) were recorded in gingival crevicular fluid. Probing depth, clinical attachment level, gingival and plaque indices and bleeding on probing were also measured. The gingival crevicular fluid and clinical parameters were recorded at baseline and 6 wk after periodontal treatment. RESULTS: The study showed statistically significant improvement of clinical parameters in both smokers and nonsmokers after periodontal treatment. Moreover, the baseline IL-1ß levels were significantly higher in smokers compared with nonsmokers (p < 0.05). After periodontal treatment, the IL-1ß levels were significantly reduced in both smokers and nonsmokers (p < 0.05). There were no significant differences in TOS and TAS between periodontitis patients and healthy controls at baseline and 6 wk after periodontal treatment. The level of IL-1ß in gingival crevicular fluid was positively correlated with TOS in both smokers and nonsmokers. CONCLUSIONS: Periodontal treatment improved the clinical parameters in both smokers and nonsmokers. The results confirm that periodontal therapy has an effect on IL-1ß levels in gingival crevicular fluid, but not on TOS and TAS.

Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves.

The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.

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.

[Phenothiazines are slowly oxidizable substrates of horseradish peroxidase].

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. k(cat) and K(m) values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH>5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.

Euphorbia peroxidase catalyzes thiocyanate oxidation in two different ways, the distal calcium ion playing an essential role.

The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.