Labeled sandwich-type electrochemical immunosensor based on Ti3C2Tx/AuNP and Ti3C2Tx/HKUST-1/TB composites for early liver cancer detection.

A novel sandwich-type electrochemical immunosensor for the detection of the liver cancer marker alpha-fetoprotein (AFP) in human serum is proposed. The two-dimensional MXene material Ti3C2Tx was first prepared using etching and ultrasonic stripping, and then Ti3C2Tx was used to reduce chloroauric acid to form Ti3C2Tx/AuNP composites which were modified on the surface of the glassy carbon electrodes to form probe-type sensors. The Ti3C2Tx/AuNPs provide a large number of binding sites for the AFP capture antibody (Ab1) and increase the electrochemical reaction active site. The Ti3C2Tx/copper metal-organic frameworks HKUST-1 composite was also prepared by solvothermal method and combined with toluidine blue (TB) and AFP detection antibody (Ab2) to form a labeled sandwich-type electrochemical immunosensor. The sensor achieved trace detection of AFP from 0.1 to 100 ng/mL with a detection limit of 0.073 pg/mL and possesses good selectivity, stability, and reproducibility. The sensor performs well in clinical samples and has good potential for clinical applications.

Performance comparison of photodynamic antimicrobial chemotherapy with visible-light-activated organic dyes: Rose bengal, crystal violet, methylene blue, and toluidine blue O.

This study evaluated the photobiocidal performance of four widely distributed visible-light-activated (VLA) dyes against two bacteria (Staphylococcus epidermidis and Escherichia coli) and two bacteriophages (phages MS2 and phi 6): rose bengal (RB), crystal violet, methylene blue, and toluidine blue O (TBO). The photobiocidal performance of each dye depended on the relationship between the type of dye and microorganism. Gram-negative E. coli and the non-enveloped structure of phage MS2 showed more resistance to the photobiocidal reaction than Gram-positive S. epidermidis and the enveloped structure of phage phi 6. RB had the highest potential to yield reactive oxygen species. However, the photobiocidal performance of RB was dependent on the magnitude of the surface charge of the microorganisms; for example, anionic RB induced a negative surface charge and thus electrical repulsion. On the other hand, the photobiocidal performance of TBO was observed to be less affected by the microorganism type. The comparative results presented in our study have significant implications for selecting photodynamic antimicrobial chemotherapy (PACT) dyes suitable for specific situations and purposes. Furthermore, they contribute to the advancement of PACT-related technologies by enhancing their applicability and scalability.

Chemical binding of molecular-imprinted polymer to biotinilated antibody: Utilization of molecular imprinting polymer as intelligent synthetic biomaterials toward recognition of carcinoma embryonic antigen in human plasma sample.

In this study, a novel biosensor based on molecular imprinting polymer (MIP) methodology was fabricated toward recognition of carcinoembryonic antigen (CEA). For this purpose, poly (toluidine blue) (PTB) was electropolymerized on the surface of gold electrode in the absence and presence of CEA. So, the target molecules were entrapped into the imprinted specific cavities of MIP. Obtained results show that, the binding affinity of the MIP system was significantly higher than that of revealed for the nonimprinted polymer (NIP) system, MIP-based biosensor revealed linear response from (0.005 to 75 µg/L) and low limit of quantification of (0.005 µg/L) by using chronoamperometry technique, leading to CEA monitoring in real and clinical samples. Thus, a novel technique for rapid, simple, sensitive and affordable monitoring of CEA (LLOQ = 0.005 µg/L) has provided through developed biosensor. From a future perspective, moreover, this method can be considered as an applicable candidate in biomedical and clinical analysis for point-of-care usages.

Enzymatic recognition of hydrogen peroxide (H2 O2 ) in human plasma samples using HRP immobilized on the surface of poly(arginine-toluidine blue)- Fe3 O4 nanoparticles modified polydopamine; A novel biosensor.

In this study, an innovative strategy was proposed for the electrocatalytical reduction and enzymatic biosensing of hydrogen peroxide (H2 O2 ) using chronoamperometry technique. For the first time, immobilization of horseradish peroxidase (HRP) in polydopamine-modified magnetic nanoparticles (PDA-MNPs) was successfully performed. Also, poly(l-arginine/toluidine blue) film-modified glassy carbon electrode was constructed through co-electropolymerization of l-arginine and toluidine blue on the surface of GCE using cyclic voltammetry technique. The engineered hybrid thin film provides strong functionalities for efficient grafting of PDA-MNPs which, in turn, enable the covalent immobilization of HRP. The proposed biosensor was used for the detection of H2 O2 in the range of 0.5-30 µM with a low limit of quantification 0.23 µM. It also was successfully applied for the investigation of hydrogen peroxide in human plasma samples.

Mast cells and collagen fibrillogenesis.

This article presents 20 combinations of histochemical stainings for the determination of mast cell co-localization with the fibrous component of the connective tissue in the fibrillogenesis course. Best results were obtained using metachromatic detection of mast cells in combination with silver or picro-fuchsin impregnation, staining with brilliant green using van Gieson staining, and a combination of aniline blue staining with neutral red. Proposed variants of histochemical protocols open up new opportunities to analyze the participation of mast cells in extracellular matrix remodeling of the tissue microenvironment in the course of adaptive and pathological processes. Results obtained expand the current theoretical views of the process of fibrillogenesis in the extracellular matrix. They also shed new light on the participation of mast cell secretion components in the molecular mechanisms of fiber formation.

Toluidine blue versus frozen section for assessment of mucosal tumor margins in oral squamous cell carcinoma.

BACKGROUND: When the resected specimen is sent for intraoperative margin assessment, all margins are grossly checked, and selected margins undergo a frozen section (FS) examination. Therefore, there is a possibility of sampling error. This study evaluated the effectiveness of using toluidine blue (TB) as an intraoperative triage screening tool to detect positive mucosal margins of the resected specimens of oral squamous cell carcinoma (OSCC) and serve as a guide for FS sampling. METHODS: Surgical samples of 30 consecutive patients with biopsy-proven OSCC were included in the study. A total of 140 mucosal margins were analyzed intraoperatively by TB and FS, the results were compared with the final histopathology. RESULTS: Of the 140 examined mucosal tumor margins, 14 stained positives with TB, six were true-positives, eight were false-positives, and there were no false-negatives, as confirmed by final histopathology of the same margins. The diagnostic performance measures were sensitivity 100.0%; specificity 94.0%; positive predictive value (PPV) 42.9%; negative predictive value (NPV) 100.0%; and accuracy 94.3% (95% CI: 89.0-97.5%). For FS, there were three true-positives, three false-negatives, and no false-positives. The diagnostic performance measures were sensitivity 50.0%; specificity 100.0%; PPV 100.0%; NPV 97.8%; and accuracy 97.9% (95% CI: 93.9-99.6%). CONCLUSION: TB is less specific but more sensitive than FS for detecting positive mucosal margins of resected OSCC. Screening the tumor mucosal margins with TB before FS sampling may help identify more tumor-bearing margins. TRIAL REGISTRATION: This trial was registered at ClinicalTrials.gov. Registration number: NCT03554967 . Registration date: June 13, 2018.

Novel ratiometric electrochemical sensor for no-wash detection of fluorene-9-bisphenol based on combining CoN nanoarrays with molecularly imprinted polymers.

No-wash detection of small molecules in real samples has been attracting attention in the field of sensors including electroanalytical biosensors. Based on the direct electrochemical oxidation of fluorene-9-bisphenol (BHPF) on a CoN nanoarray electrode, we developed a ratiometric molecularly imprinted polymeric electrochemical (MIP-EC) sensor to realize no-wash detection of BHPF in serum and tap water. The CoN nanoarray in situ grown on carbon cloth (CC) served as the working electrode, which could load the electroactive toluidine blue (TB) and be modified by the MIPs. As the MIP concentration on the modified electrode surface was increased, the amount of BHPF exposed on the electrode surface increased and the amount of exposed TB decreased. Thus, the values of ΔITB and ΔIBHPF decreased and increased, respectively, with an increasing amount of BHPF. Therefore, a ratiometric strategy was established by using the value of ΔITB/ΔIBHPF as the instruction response to realize detection of BHPF with high sensitivity and reliability. The developed ratiometric MIP-EC sensor showed strong anti-interference ability, good detection reproducibility and stability towards no-wash detection of BHPF as shown from tests with real samples. This work can further provide theoretical and practical guidance for the detection of other familiar small molecules.

Anti-Trypanosoma cruzi effect of the photodynamic antiparasitic chemotherapy using phenothiazine derivatives as photosensitizers.

Chagas disease is endemic in Latin America and increasingly found in non-endemic countries. Its treatment is limited due to the variable efficacy and several side effects of benznidazole. Photodynamic antimicrobial chemotherapy (PACT) may be an attractive approach for treating Chagas disease. Here, the trypanocidal activity of PACT was investigated in vitro using phenothiazine derivatives. The cytotoxicity of both, methylene blue (MB) and toluidine blue (TBO), was determined on macrophages cultures using AlamarBlue method. The trypanocidal activity of the two photosensitizers was initially evaluated by determining their IC50 values against trypomastigote forms. After this, the trypanocidal effect was evaluated in cultures of infected macrophages using an automatized image analysis protocol. All experiments were performed in the dark and in the clear phase (after a photodynamic exposure). The compounds showed no cytotoxicity in both phases at the tested concentrations. The IC50 values for the sole use of MB and TBO were 2.6 and 1.2 µM, respectively. The photoactivation of the compounds using a fixed energy density (J/cm2) caused a reduction of the IC50 values to 1.0 and 0.9 µM, respectively. It was found that, on infected macrophage, the use of TBO significantly reduced the number of infected cells and parasitic load, and this effect was increased in the presence of light. The results of the present study are indicative that PACT may be considered as both selective and effective therapeutic intervention for treating Chagas disease.

Target-triggered configuration change of DNA tetrahedron for SERS assay of microRNA 122.

A surface-enhanced Raman scattering (SERS) method is proposed for the assay of microRNA 122 based on configuration change of DNA tetrahedron. Firstly, a DNA tetrahedron was self-assembled with one vertex labeled with toluidine blue (TB). Then, it was immobilized on the porous Ni/SiO2@PEI@Au as a SERS platform, which was characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). At this time, the DNA tetrahedron was contracted; so, the TB is close to AuNPs and the Raman signal is high. When target microRNA 122 existed, with the nicking enzyme amplification strategy, a great deal of DNA signal chains (S5) was obtained, which can extend the contracted DNA tetrahedron and change it into a three-dimensional DNA tetrahedron. In this case, the TB was far from AuNPs, resulting in a lower Raman signal. Due to the configuration change of DNA tetrahedron, the Raman signal at 1624 cm-1 (with the excitation wavelength of 633 nm) has a linear relationship with the logarithm concentration of microRNA 122. This SERS assay has high sensitivity for microRNA 122 with a determination range from 0.01 aM to 10 fM and a detection limit of 0.009 aM. The recoveries from spiked samples were in the range 95 to 109%. This SERS strategy is designed based on the target-triggered configuration change of DNA tetrahedron, which can give new insight for DNA structures in bioanalysis. Graphical abstract A sensitive surface-enhanced Raman scattering (SERS) biosensor was developed to detect microRNA 122 using the configuration change of DNA tetrahedron to indirectly control the position of TB and hot spot.

Development of choline biosensor using toluidine blue O as mediator.

A new choline oxidase (ChO) and toluidine blue O (TBO) based amperometric choline biosensor was reported in this article. An amperometric choline biosensor with immobilization of TBO (as a mediator), ChO onto polypyrrole-polyvinylsulphonate (PPy-PVS) film was accomplished on the surface of a platinum electrode. ChO was immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA). TBO was used as the mediator. Choline is oxidized to betaine and hydrogen peroxide in an oxygenated environment by ChO. Mediator reduced by reaction with hydrogen peroxide. The amperometric response was based upon the electrocatalytic properties of TBO. Optimum pH and temperature values were 7.0 and 30 °C, respectively. There was linearity between 1.0 × 10-8 and 2.0 × 10-8 M (R2 = 0.9805). The detection limit of the biosensor was 1.0 × 10-9 M and response time of the biosensor was 200 s. The storage stability and reproducibility of the biosensor were also investigated. Interfering effect of several interferants such as ascorbic acid, uric acid, alanine, dopamine, paracetamol, cysteine, and glucose on the choline biosensor was examined. The developed biosensor was tested in determinations of content in a synthetic blood sample.

Effect of Bone Marrow Autotransplantation on the Numerical Population of Tryptase-Positive Mast Cells, Heparin Sulfation Degree, and Histamine Content.

We studied the effect of bone marrow autotransplantation on the morphofunctional properties and numerical population of mast cells. The experiments were performed on 4-monthold male mice. The animals received an injection of a suspension of bone marrow obtained from the femoral epiphyses of these animals into the caudal vein. In 40 min and 2 h after autotransplantation, the number of tryptase-positive mast cells increased by 1.1 times. The formation of groups of mast cells near erythroid-neutrophil islets and near blood vessels was observed. The proportion of metachromatic mast cells significantly increased. By the degree of mast cells degranulation, we detected non-degranulated up to 48.0±1.4% (vs 55.2±1.2% in intact mice) and moderately degranulated mast cells 22.0±1.2% (vs 18.2±0.9% in intact mice); the percentage of actively degranulated cells was 10.0±0.8% (vs 3.6±0.9% in intact mice; p<0.05). Morphometric parameters of mast cells were changed, with a slight increase in their diameter and distance between the cells. The number of histamine-containing mast cells increased significantly (by 3.2 times in 40 min and by 5.9 times in 2 h) and histamine content in these cells also increased. Thus, bone marrow autotransplantation led to intensification of degranulation and sulfation of mast cells and the release of histamine from them.

Gray-level co-occurrence matrix analysis of chromatin architecture in periportal and perivenous hepatocytes.

Periportal hepatocytes (PPHs) and perivenous hepatocytes (PVHs) in standard optical microscopy appear to be morphologically identical. However, the functional properties of these two cell populations and their roles in liver lobules are not the same. Despite significant differences in gene expression between these two hepatocyte populations, it is still unclear whether the differences are present at the higher levels of chromatin organization. In this study, we present results, indicating that periportal and perivenous hepatocytes, when stained using toluidine blue histological dye, have different chromatin textural patterns quantified with gray-level co-occurrence matrix (GLCM) method. Hepatic tissue was obtained from ten male, healthy mice. Chromatin structures were analyzed using GLCM. For each structure, we measured the values of angular second moment, inverse difference moment, GLCM Contrast, GLCM Variance, and GLCM Sum Variance. The results indicate that there is a statistically significant difference in all GLCM mathematical parameters except the contrast. In addition, some chromatin GLCM features were in correlation with serum aminotransferase levels in perivenous, but not in periportal hepatocytes. To the best of our knowledge, this is the first study to test the nuclear morphological differences between hepatocytes using GLCM and to investigate the respective relation with serum liver enzymes.

Role of Toluidine Blue-O Binding Mechanism for Photooxidation in Bioinspired Bacterial Membranes.

The alarming increase in bacterial resistance to antibiotics has demanded new strategies for microbial inactivation, which include photodynamic therapy whose activity relies on the photoreaction damage to the microorganism membrane. Herein, the binding mechanisms of the photosensitizer toluidine blue-O (TBO) on simplified models of bacterial membrane with Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) were correlated to the effects of the photoinduced lipid oxidation. Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were also used as a reference of mammalian membranes. The surface pressure isotherms combined with polarization-modulated infrared reflection absorption spectroscopy revealed that TBO expands DOPC, DOPE, and DOPG monolayers owing to electrostatic interactions with the negatively charged groups in the phospholipids, with a stronger adsorption on DOPG, which has a net surface charge. Light irradiation made the TBO-containing DOPC and DOPE monolayers less unstable as a result of the singlet oxygen (1O2) reaction with the chain unsaturation and hydroperoxide formation. In contrast, the decreased stability of the irradiated TBO-containing DOPG monolayer suggests the cleavage of carbon chains. The anionic nature of DOPG allowed a deeper penetration of TBO into the chain region, favoring contact-dependent reactions between the excited triplet state of TBO and lipid unsaturations or/and hydroperoxide groups, which is the key for the cleavage reactions and further membrane permeabilization.

Topical Aluminum Chloride and Monsel's Solution Block Toluidine Blue Staining in Mohs Frozen Sections: Mechanism and Solution.

BACKGROUND: A diminished-staining artifact is observed in some Mohs frozen sections that are stained in toluidine blue (T-blue). Such an artifact, not yet described in the literature, may interfere with a Mohs surgeon's accurate reading. The authors hypothesize that topical hemostatic agents, aluminum chloride, and Monsel's solution are the causative factors. OBJECTIVE: To evaluate the aforementioned topical hemostatic agents as a potential cause of the nonstaining artifact, to propose the mechanism associated with this phenomenon, and to develop a method to prevent or rectify the problem. MATERIALS AND METHODS: Leftover Mohs frozen sections and specimens were treated with aluminum chloride or Monsel's solution and processed with routine Mohs histology. RESULTS: Nonstaining artifact is reproduced in aluminum chloride or Monsel's solution-treated ex vivo skin specimens. The authors found that ethylenediaminetetraacetic acid (EDTA), a chelating agent, can reverse the staining blockage. Such a finding suggests that aluminum or ferric cations bind to tissue and subsequently inhibit T-blue from interacting with the tissue. Direct binding of ferric cations to the tissue section is demonstrated with Prussian blue iron staining. CONCLUSION: By rinsing Mohs frozen sections in an EDTA solution before T-blue staining, the authors could prevent hemostatic agent-induced nonstaining. Applying an EDTA wash and restaining the slides can correct the same artifact.

Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model.

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno-inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.

Dog sperm DNA: Raw semen evaluation with Toluidine blue stain.

The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty-one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue-violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min) for any of the TB patterns. All DTT incubation times (2, 5 and 30 min) showed 100% sperm positive to TB. To conclude, it was possible to simplify the TB stain and determine the different patterns in canine spermatozoa. Also, DTT can be used both as a positive control for the stain and to evaluate individual susceptibility to decondensation in vitro.

Simultaneous voltammetric immunodetection of alpha-fetoprotein and glypican-3 using a glassy carbon electrode modified with magnetite-conjugated dendrimers.

The authors describe an electrochemical immunoassay for simultaneous determination of alpha-fetoprotein (AFP) and glypican-3 (GPC-3) which are important biomarkers for early detection of hepatocellular carcinoma (HCC). Magnetite (Fe3O4) nanoparticles (NPs) were decorated with hyperbranched amino functionalized dendrimers. The modified NPs were coupled to the antibodies against AFP and GPC-3. The electrochemical behaviour of the Fe3O4 NPs and dendrimer-modified NPs were studied. A glassy carbon electrode was then modified with the NP-conjugated antibodies and biomolecular interactions were studied by using cyclic voltammetry and electrochemical impedance spectroscopy. Dual differential pulse voltammetric sensing was performed by utilizing the redox probes; Prussian blue for AFP and toluidine blue for GPC-3. The biomarkers can be detected best at voltages of 0.25 mV and - 0.54 mV (vs Ag/AgCl) for AFP and GPC-3, respectively. The low working potentials makes the method more selective over other electroactive species present in real human serum samples. Response is linear in 0.02 to 10 ng mL-1 concentration ranges of both AFP and GPC-3; and the respective detection limits are 50 and 70 pg mL-1. The method was validated by analysing spiked human serum samples. In our perception, the method is of great clinical significance as combination of GPC-3 and AFP increases the sensitivity of detection of HCC. Graphical abstract Schematic presentation of polyamidoamine (PAMAM) dendrimers modified magnetite (Fe3O4) nanoparticles as electrochemical sensing platform using redox dyes Prussian blue and toluidine blue for simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC-3), respectively by differential pulse voltammetry.

Real-Time Visualization of Lysosome Destruction Using a Photosensitive Toluidine Blue Nanogel.

Breaking the lysosome helps its sequestered payloads access their molecular targets in cells and thus enhances the intracellular drug delivery. Current strategies for lysosomal escape involve direct physical interactions with the lipid membrane. These interactions pose a systemic toxicity and uncontrolled membrane rupture risk. Here, we report a light-detonated lysosome disruption using a hyaluronan (HA) nanogel packed with toludine blue (TB). The HA/TB nanogel is concentrated within the lysosomes. The applied light assists TB in generating reactive oxygen species and destroying the lysosome in situ, both in cells and isolated lysosomes. Real time fluorescent tracking reveals that quenched TB fluorescence recovers along with lysosome explosion, relocates to the nucleus, and is presented as a fluorescent sparkling in cells. This HA/TB, composed of all clinically approved materials, represents a biocompatible and facile strategy to "bomb" lysosomes in a spatiotemporally controlled fashion.

A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species.

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high-precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue-stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.

Decolorization of dyes by a novel sodium azide-resistant spore laccase from a halotolerant bacterium, Bacillus safensis sp. strain S31.

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v-1) methanol, ethanol, and acetone. In contrast to many other laccases, NaN3, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l-1) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.