Use Of Tetramethylbenzidine Research Articles

FeTOMPP with Peroxidase-Like Activity as Peroxidase Mimics for Colorimetric Sensing of H2O2 and Ethanol

In this study, FeTOMPP (Tetrakis(4-methoxyphenyl) phenylporphyrinatoiron(III)) was synthesized and investigated for its peroxidase-like activity. The catalytic performance of FeTOMPP as a peroxidase mimic was explored by utilizing the colorless peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB), in the presence of hydrogen peroxide (H2O2). The oxidation of TMB by FeTOMPP resulted in the formation of its blue oxidized state. This enzymatic reaction was employed for the quantitative detection of H2O2, with a linear detection range spanning from 1.0 to 100 µM. The detection limit achieved using FeTOMPP as the catalyst was determined to be 2.4 µM. Furthermore, kinetic studies were conducted to investigate the interaction between FeTOMPP and H2O2. The results indicated that FeTOMPP exhibited favorable affinity towards H2O2, further highlighting its potential as a peroxidase mimic. In addition to its peroxidase-like activity, a simple and sensitive visual and colorimetric method was developed for the detection of ethanol by combining FeTOMPP with alcohol oxidase. The method relied on the use of TMB as the substrate. Ethanol-containing samples were subjected to the enzymatic reaction, resulting in the generation of a color change. The detection of ethanol was achieved with a minimum detection limit of 0.95 % v/v, and the linear range for quantification extended from 0 to 15 % v/v. This study demonstrates the potential application of FeTOMPP as a peroxidase mimic for the sensitive detection of H2O2 and the development of a colorimetric method for ethanol detection. The findings highlight the versatility and promising capabilities of FeTOMPP in various analytical and diagnostic applications. HIGHLIGHTS The potential application of FeTOMPP as a peroxidase mimic for the sensitive detection of H2O2. The development of colorimetric method for ethanol detection by combining FeTOMPP with alcohol oxidase. GRAPHICAL ABSTRACT

A naked-eye liquid-phase colorimetric assay of simultaneous detect cysteine and lysine

In this work, we proposed a naked-eye colorimetric strategy for simultaneous detection of cysteine and lysine based on 3,3′,5,5′-tetramethylbenzidine (TMB) system in a neutral condition (pH 7.0). Based of the fact that Ag+ could oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidized TMB (oxTMB) and induce a blue color solution corresponding to an absorption peak centered at 652 nm, thus the adding of cysteine or lysine to formation of different product results in the blue color fading to colorless quickly or change to scarlet, respectively. With this strategy, the limit of detection in experimental measurement for cysteine and lysine is 2.46 μM and 3.87 μM, respectively. And the selectivity can also be guaranteed among the other amino acid. The cost is quite low since the use of TMB and Ag+. More importantly, the colorimetric detection operation is very simple without any further modification process and the reaction under physiological conditions is more meaningful for detection of amino acid.

Use of 3,3',5,5' tetramethylbenzidine as new electrochemical indicator of DNA hybridization and its application in genossensor

Electrochemical tools are important biosensor platforms for disease diagnosis, due to their speediness, easiness, low cost and portability. However, for DNA detection, the use of indicators and/or intercalators is necessary to improve electrochemical sensitivity.Currently, ethidium bromide (EthBr) is the cheapest and most used DNA intercalators, but presents carcinogenic and teratogenic properties. Other indicators may be important for DNA photonic detection, and besides being more expensive, they behave similarly to EthBr.This investigation shows for the first time the use of tetramethylbenzidine(TMB) as a new remarkable non-carcinogenic DNA indicator for genosensing purposes, which may be used for nucleic acid detection of microorganisms, based on complementarity of base-pairing between probe and target molecules.The results indicate that TMB can be used as a new electrochemical indicator readily applicable in genosensors, which is able to detect the hybridization of single stranded DNA probe with its complementary target strand. An additional advantage of TMB, beside its non-genotoxicity, is the electrochemical reduction property, which prevents interference of serum components and other oxidative samples in the electrochemical analysis.

Comparative study of amplification systems in immunoenzyme assays for the diagnosis of American tegumentary leishmaniasis

We compared the accuracy and reliability of three amplification systems for enzyme immunoassays in the detection of specific IgG antibodies for the diagnosis of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in patients from an endemic area in Rio de Janeiro, Brazil. Partially soluble antigens obtained from the promastigote forms of L. (V.) braziliensis were used. For development of the reaction, two chromogens, 1,2-orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB), and a fluorogen, 4-methylumbelliferylphosphate (MUP), were tested. The performance of each system was compared using the following parameters: accuracy, intraclass correlation coefficient (ICC), and area under the receiver operating characteristic (ROC) curve. Sensitivity was the same (97.4%) for all systems. The reliability was excellent (ICC = 98.6, 98.7, and 99.1%) and specificity was 93.7, 95.8, and 97.4% for OPD, MUP, and TMB, respectively, showing no statistical significance. Despite the absence of differences in the performance of the three systems, the use of TMB is suggested because of its operational advantages, such as low cost compared with fluorogens, easy manipulation, greater stability, and lower toxicity.

Use of tetramethylbenzidine for the spectrophotometric sequential injection determination of free chlorine in waters

A sequential injection (SI) method was developed for the spectrophotometric determination of chlorine based on the reaction between tetramethylbenzidine (TMB) and free chlorine. The advantages resulting from the use of TMB are considerable: TMB is highly selective for chlorine, it enables a fairly low quantification limit and represents a less toxic alternative to reagents such as tolidine. The use of this reaction in SI adds other advantages as it enhances the degree of automation, minimisation of reagent consumption (6.8 μg TMB/assay) and low effluent production (2.5 mL/determination). The developed method allowed a quantification limit of 90 μg/L with a working range of 0.09–1.30 mg OCl −/L and a determination rate of 60 det./h. Based on these features, the system was applied to tap-water and surface water samples with no previous treatment required. The results obtained with the developed system were compared to the reference method, diethyl- p-phenylelediamine (DPD) colorimetric method, and proved not to be statistically different.

Comparison of wheat germ agglutinin–horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury

Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin–horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer is administered in dried form. An alternative method is described for anterograde labelling of cortical motor neurons using biotinylated dextran. This tracer may be applied by either microinjection of 10% biotinylated dextran or implanting small globules of the dried tracer into the motor cortex. While more laborious, microinjection results in better anterograde labelling than implantation of dried biotinylated dextran. A procedure is also described for preparing serial coronal sections through the entire spinal cord and thaw-mounted on a minimum number of slides. The labelled nerve processes in these tissue sections can be visualised in the spinal cord under a fluorescent microscope following incubation with cy3–streptavidin complex. Permanent labelling of the biotinylated nerve processes is achieved by incubation of tissue sections with streptavidin–horseradish peroxidase conjugate followed by stringent washes and staining with tetramethylbenzidine. Use of tetramethylbenzidine allows resolution of a greater number of finer labelled processes than diaminobenzindine and allows clear visualisation of individual regenerating corticospinal tract processes. Using these procedures, we demonstrate that the corticospinal tract is completely lesioned by a standardised contusion spinal cord injury produced by the New York University weight-drop device.

Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue

Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.

A combined pre- and post-embedding double labeling in the central nervous system with good tissue preservation

Multiple labeling at the electron microscopic level is routinely done in various parts of the central nervous system. We demonstrate that the pre-embedding tetramethylbenzidine (TMB) reaction for visualizing horseradish peroxidase (HRP) of Olucha and the slow osmication of Henry combined with a post-embedding nonetching immunogold method will also preserve good ultrastructure. Furthermore, the post-embedding immunocytochemistry of some neurotransmitters, i.e. gammaaminobutyric acid (GABA), can be done months after the tissue has been reacted for HRP and embedded in regular epon.Pre-embedding histochemistry:The use of TMB as a chromagen for the demonstration of neuronally transported HRP has both the advantage of being highly sensitive and of producing very specific needle-like crystals. Olucha et al demonstrated that one could further stabilize this reaction product with amonium heptamolybdate. Unfortunately the next step, fixation with regular osmium tetroxide, often resulted in the loss of the reaction product. However, the slow osmication with a lower pH (5.5) in the phosphate buffer at room temperature as recommended by Henry et al prevented this loss, and at the same time resulted in well preserved ultrastructure.

The use of tetramethylbenzidine for solid phase immunoassays

A rapid, se nsitive, solid-phase immunoassay, using horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine, was found to be more sensitive and the colour developed was more stable compared with HRPO using 4-chloro-1-naphthol or diaminobenzidine. Assay sensitivity was equal to or greater than that obtained with the alkaline phosphatase reaction. The essential step was to incubate filters in 1% dextran sulphate for 10 min in a pH 5 (10 mM citrate-EDTA) buffer before reacting with TMB (0.05 mg/ml in the same buffer).

Evaluation of Dot-immunobinding Assay for Carcinoembryonic Antigen Determination in Nipple Discharge as an Adjunct in the Diagnosis of Early Breast Cancer

We have previously reported carcinoembryonic antigen (CEA) measurement in nipple discharge to be a useful adjunct in the diagnosis of non-palpable breast cancer. As an extension, a dot-immunobinding assay was developed to screen a large number of patients with nipple discharge for non-palpable breast cancer. The principle is as follows. CEA bound to a solid phase monoclonal anti-CEA antibody is detected by a second monoclonal anti-CEA antibody conjugated with horseradish peroxidase. The use of tetramethylbenzidine as a chromogen results in a stable color reaction that can be semiquantitively analyzed by the naked eye. The CEA levels determined by this dot assay correlated well with CEA levels determined using the former Elmotec assay. To determine whether or not the method could also be feasible in the detection of non-palpable breast cancer, a collaborative study from 12 Japanese institutes was organized. The CEA levels in nipple discharges from 155 patients were assayed. Thirteen of 30 patients with palpable breast cancer and 22 of 30 patients with non-palpable breast cancer exhibited CEA values higher than 400 ng/ml, a cut-off value determined using 89 benign controls. The specificity (91%) and sensitivity (73%) of this test were higher than those of mammography or cytology. The incidence of elevated CEA levels in nipple discharge correlated significantly with the incidence of intratumoral antigen expression. Thus, the system could prove useful in screening for early breast cancer.

Dorsal funicular projections to the dorsal column nucleus in a reptile, Caiman crocodilus.

Projections to the dorsal column nucleus were investigated in the reptile Caiman crocodilus following application of horseradish peroxidase (HRP) crystals to the cervical spinal cord after dorsal myelotomy. Brains were processed for HRP histochemistry by use of tetramethylbenzidine as the chromogen. Injection of the dorsal funiculus alone labelled axons that terminated solely in the ipsilateral dorsal column nucleus. Application of HRP that extended beyond the dorsal funiculus labelled axons that terminated in brainstem areas in addition to the dorsal column nucleus depending upon the injection site. Retrogradely labelled neurons in the brainstem were seen only after HRP injections that extended beyond the dorsal funiculus and varied depending upon the injection site. Dorsal funiculus fibers travel rostrally and turn laterally to reach more anterior portions of the dorsal column nucleus. Fibers peel off in an oblique ventrolateral fashion to terminate in the posterior part of the nucleus where boutons are intermixed with fibers. The anterior portion of the nucleus contains a greater proportion of axon terminals as compared to fibers than does the posterior part. A graphic reconstruction of the dorsal column nucleus was made by superimposing serial sections to view the nucleus in transverse, sagittal, and horizontal planes.

Variability in the distribution of callosal projection neurons in the adult rat parietal cortex

Previous reports have shown that the barrel field area of the parietal cortex of the adult rat contains relatively few callosal projection neurons, even though callosal projection neurons are abundant in this cortical region in the neonatal rat. Furthermore, it has been shown that many of the callosal neurons which ssem to disappear as the animal matures do not die, but project to ipsilateral cortical areas. These findings rely on the ability of retrograde transport techniques which utilize injections of horseradish peroxidase (HRP) or of fluorescent dyes into one hemisphere. We now show that several technical modifications of the HRP technique yield a wider distribution of HRP-containing neurons in the contralateral barrel field area of the adult rat than previously reported. These include implants of HRP pellets into transected axons of the corpus callosum, the addition of DMSO and nonidet P40 and to Sigma VI HRP, wheat germ agglutinin HRP and the use of tetramethyl benzidine as the chromogen in the reaction procedure. Our findings have implications for transport studies in general and for the development of the cortical barrel field in particular.

Brainstem projections of sensory and motor components of the vagus nerve in the rat.

The sensory and motor connections of the cervical vagus nerves and of its inferior ganglion (nodose ganglion) have been traced in the medulla and upper cervical spinal cord of 16 male Wistar rats by using horseradish peroxidase (HRP) neurohistochemistry. The use of tetramethyl benzidine (TMB) as the substrate for HRP permitted the visualization of transganglionic and retrograde transport in sensory nerve terminals and perikarya, respectively. The vagus nerve in the rat enters the medulla in numerous fascicles with points of entry covering the entire lateral aspect of the medulla extending from level +4 to -6 mm rostrocaudal to the obex. Fascicles of vagal sensory fibers enter the dorsolateral aspect of the medulla and travel to the tractus solitarius (TS) which was labeled for over 8.8 mm in the medulla. The caudal extent of the TS receiving vagal projections was found in lamina V of the cervical spinal cord (C1 to C2). Sensory terminal fields could be visualized bilaterally in the nucleus of the tractus solitarius (nTS), area postrema (ap) and dorsal motor nucleus of the vagus nerve (dmnX). The ipsilateral projection to the nTS and the dmnX was heavier than that found on the contralateral side. The area postrema was intensely labeled on both sides. Motor fibers from HRP-labeled perikarya in the dmnX travel ventromedially in a distinct fascicle and subsequently subdivide into a number of small fiber bundles that traverse the medullary reticular formation in the form of a fine network of HRP-labeled fibers. As these fibers from the dmnX approach the ventrolateral aspect of the medulla they are joined by axons from the nucleus ambiguus (nA), nucleus retroambigualis (nRA) and the retrofacial nucleus (nRF). These latter fibers form hairpin loops in the middle of the reticular formation to accompany the axons from the dmnX exiting from the medulla in a ventrolateral location. HRP-labeled perikarya, in contrast to transganglionically transported HRP in sensory terminals in the nTS, were visualized on one side only, thus indicating that motor control via the vagus nerve is exerted only by motor neurons located ipsilaterally. Sensory information on the other hand, diverges to many nuclear subgroups located on both sides of the medulla.