Application Of Horseradish Peroxidase Research Articles

Development of a lateral flow immunoassay with HRP enhancement for spiked SARS-CoV-2 protein N detection in human saliva

The Coronavirus (SARS-CoV-2) is an enveloped RNA virus that caused a dangerous COVID-19 pandemic following an outbreak in Wuhan, China in 2019. In response to the pandemic, the development of lateral flow assays (LFAs) has been crucial for the detection of viruses, commonly targeting the spike (S) or nucleocapsid (N) protein in nasopharyngeal swab (NS) specimens. COVID-19, caused by the SARS-CoV-2 virus, predominantly manifests as a respiratory tract infection-like illness, characterized by symptoms such as fever, dry cough, upper airway congestion, runny nose, sore throat, myalgia, headache, and exhaustion. This study presents the development of an LFA targeting the N protein to detect the coronavirus in saliva specimens, using a sandwich format. The use of 80 nm gold nanoparticles (AuNPs) has been investigated, and the application of horseradish peroxidase (HRP) and TMB substrate has been employed to enhance the limit of detection (LOD). The results demonstrate a significant 200-fold improvement in LOD, from 10 to 50 pg/ml, for N protein spiked in saliva samples after the application of HRP-TMB. This finding highlights an important advancement towards the utilization of saliva samples in diagnostic applications.

Visible-light-driven photo-peroxidase catalysis: high-efficiency degradation of indole in water.

The demand for H2O2 restricts the wider application of horseradish peroxidase (HRP) in degradation. In this work, a novel photoenzyme synergistic catalytic system was developed for high-efficiency degrading of indole in water by HRP without extra H2O2. The HRP was immobilized on CN-ZIF prepared by the combination of g-C3N4 and ZIF-8 to achieve photo-peroxidase catalyst HRP/Zn-CN-ZIF. Under visible light, photogenerated electrons and H2O2 from HRP/Zn-CN-ZIF participated in the biocatalytic cycle of HRP directly. As a result, the indole at 20 mg L-1 in water was degraded completely in 2 h by the HRP/Zn-CN-ZIF photoenzyme synergistic catalytic system without the addition of H2O2. Furthermore, HRP/Zn-CN-ZIF exhibited superior visible light absorption and charge transfer ability compared to g-C3N4. The results of the mechanism studies suggest that ·OH would play the most significant role from the HRP/Zn-CN-ZIF in indole degradation. This research provides an efficient approach for the removal of indole from water environments.

Cassava Root Cortex Peroxidase (CCP) as a Potential Alternative Source of Peroxidase

AbstractCassava is an important starch crop in the world. Starch factories normally generate a huge amount of cassava root residual which may cause pollution to the environment. In order to find some extra uses for the root cortex, cassava root cortex peroxidase (CCP) is found in quantities up to 20 mg kg−1 in deteriorated cortex tissueand demonstrates some applications similar to horseradish peroxidase (HRP). The major native CCP is a 105‐kDa dimeric peroxidase with two 54‐kDa monomers. CCP is found to be tolerant to a broad pH range from 3 to 11 with maximum activity at pH 5.0 and maintains wide temperature range activity from 20 to 70 °C with an optimum at 65 °C. This indicates CCP to be one of the most robust peroxidases. Despite simple purification with ammonium sulfate precipitation, partial purified CCP is capable of determining glucose concentrations with glucose oxidase similar capability to HRP. For application as a reporter enzyme in immunoassays, the lab‐made secondary antibody conjugated with CCPsuccessfully detects the specific antigen in Western blot analysis using achemiluminescent substrate in the same way as HRP. From the properties and results of trial applications, CCP can be equally replaced in all applications of HRP. The enzyme should be found more potential applications to gain the acceptance. Since raw material for CCP is industrially massive, it should emphasize not only optimization of purification and process cost for industry, but also commercializeto the market.

A novel immobilized horseradish peroxidase platform driven by visible light for the complete mineralization of sulfadiazine in water

A novel immobilized enzyme driven by visible light was prepared and used for complete mineralization of antibiotics in water bodies. The immobilized enzyme was composed of carbon nitride modified by biochar (C/CN) and horseradish peroxidase (HRP), establishing the photo-enzyme coupling system with synergistic effect. Among them, the introduction of biochar not only improves the stability and loading capacity of the enzyme, but also improves the light absorption capacity and carrier separation efficiency of the photocatalyst. After the optimization of immobilization process, the solid load of HRP could reach 251.03 mg/g, and 85.03 % enzyme activity was retained after 18 days of storage at 4 °C. In the sulfadiazine (SDZ) degradation experiment, the degradation rate of HRP/C3/CN reached 71.21 % within 60 min, which was much higher than that of HRP (2.33 %), CN (49.78 %) and C3/CN (58.85 %). In addition, under the degradation of HRP/C/CN, the total organic carbon (TOC) removal rate of SDZ reached 53.14 %, which was 6.47 and 1.74 times that of CN and C3/CN, respectively. This study shows that the introduction of biochar is of great significance to the photo-enzyme cascade coupling system and provides a new strategy for the application of HRP&g-C3N4 system in wastewater treatment.

Ferric heme b in aqueous micellar and vesicular systems: state-of-the-art and challenges.

Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in monomeric form in a hydrophobic pocket containing among other amino acid side chains the two imidazoyl groups of His170 and His42. Both amino acids are important for the peroxidase activity of HRP as an axial ligand of hemin (proximal His170) and as an acid/base catalyst (distal His42). A key feature of the peroxidase mechanism of HRP is the initial formation of compound I under heterolytic cleavage of added hydrogen peroxide as a terminal oxidant. Investigations of free hemin dispersed in aqueous solution showed that different types of hemin dimers can form, depending on the experimental conditions, possibly resulting in hemin crystallization. Although it has been recognized already in the 1970s that hemin aggregation can be prevented in aqueous solution by using micelle-forming amphiphiles, it remains a challenge to prepare hemin-containing micellar and vesicular systems with peroxidase-like activities. Such systems are of interest as cheap HRP-mimicking catalysts for analytical and synthetic applications. Some of the key concepts on which research in this fascinating and interdisciplinary field is based are summarized, along with major accomplishments and possible directions for further improvement. A systematic analysis of the physico-chemical properties of hemin in aqueous micellar solutions and vesicular dispersions must be combined with a reliable evaluation of its catalytic activity. Future studies should show how well the molecular complexity around hemin in HRP can be mimicked by using micelles or vesicles. Because of the importance of heme b in virtually all biological systems and the fact that porphyrins and hemes can be obtained under potentially prebiotic conditions, ideas exist about the possible role of heme-containing micellar and vesicular systems in prebiotic times.

Interaction of indole-3-acetic acid with horseradish peroxidase as a potential anticancer agent: from docking to molecular dynamics simulation

The oxidation process, catalyzed by the peroxidase enzymes, occurs in all domains of life to detoxify the hydrogen peroxide toxicity. The most well-known, applicable and vastly studied member of the peroxidases family is horseradish peroxidase (HRP), especially the isoenzyme C (HRP C). HRP (primarily HRP C) is commercially available and applicable in biotechnology and diagnosis. Recently, a novel application of HRP has been introduced in cancer therapy as the combination of HRP with indole-3-acetic acid (IAA). The anticancer activity of HRP/IAA complex is through oxidation of IAA by HRP in hypoxic tumor condition, which leads to apoptosis and cancerous cell death. However, the molecular interaction of HRP/IAA has not been elucidated. Identifying the interaction of IAA with HRP would provide a better insight into its function and applications. In this study, molecular docking and molecular dynamics (MD) simulation were applied to determine the molecular interaction of the IAA/HRP complex. The docking study represented that IAA bound at the ‘exposed’ heme edge of the HRP enzyme, and the IAA entrance to the enzyme was situated at the carboxymethyl side-chain of the selected structure. Our computational results showed the HRP/IAA complex structure stability. While hydrogen bond formation with ARG38 and HIS42 stabilized the substrate, hydrophobic interactions with Phe68, Gly69, Leu138, Pro139, Pro141 and Phe179 contributed to IAA/HRP complex stability. The results can help to better understand peroxidase enzyme activity and would pave the way for future development of new therapeutics with improved anticancer efficacy. Communicated by Ramaswamy H. Sarma

Histo-ELISA technique for quantification and localization of tissue components

A novel Histo-ELISA technique is intended to facilitate quantification of target tissue proteins in a tissue section and involves the selection of target regions in the tissue section, application of streptavidin-conjugated HRP (horseradish peroxidase), coupled with peroxidase substrate—TMB (3,3′,5,5′-tetramethylbenzidine), and staining dye evaluation with ELISA reader. The target protein content (weight per volume unit) was translated from optical densities by a reference standard curve, obtained via parallel staining of the targeted protein-coated slides. To validate the technique, we carried out quantifications of IgG extravasation in ischemic and nonischemic brain sections in a mouse stroke model. With those obtained data and the reference of immunohistochemistry scores assessed on the adjacent sections, accuracy, sensitivity, and precision for the technique were evaluated. For all evaluated parameters, Histo-ELISA performance was either comparable to or better than the standard immunohistochemistry. A comparison with the data from the repeated measurements yielded a rather low coefficient of variation. The results confirmed that the technique is a fairly reliable quantitative test with rather high sensitivity, accuracy, precision, and reproducibility for detecting target protein content in tissue sections and that its tissue distribution and related subsequent morphological changes can be observed at the same time.

PAMAM Dendrimer Modified Reduced Graphene Oxide Postfunctionalized by Horseradish Peroxidase for Biosensing H2O2.

In this chapter, we describe the tethering of horseradish peroxidase (HRP) to reduced graphene oxide (RGO) for sensing H2O2 in serum. To accomplish this, RGO was synthesized through a green route by reducing graphene oxide (GO) prepared by Hummers method with carrot extract. The RGO was then covalently functionalized by electrochemical amination using fourth generation, amine-terminated PAMAM dendrimers. Subsequently, HRP was postfunctionalized through glutaraldehyde linkage. The synthesized RGO and the functionalization steps were well characterized by spectroscopic, microscopic, and electrochemical techniques. The application of HRP tethered RGO was demonstrated for H2O2 sensing in blood serum. This work provides scope for extending this functionalization strategy for other carbonaceous materials as well.

Application of horseradish peroxidase modified nanostructured Au thin films for the amperometric detection of 4-chlorophenol

A facile electrodeposition method was used to fabricate nanostructured Au thin films. Construction of the amperometric biosensors for 4-chlorophenol (4-CP) is based on the immobilization of horseradish peroxidase (HRP) on the Au thin films via a simple method. The as-prepared nano-Au films provide a favorable microenvironment for the immobilization of HRP and the retention of its activity. With H2O2 as oxidizing co-substrate, the HRP modified electrode displays high catalytic activity toward 4-CP. Besides, it has been demonstrated that the immobilization of HRP has a significant positive effect on the anti-fouling performance of the electrode material. Furthermore, the enzyme modified electrode was used as a bioelectrochemical sensor of 4-CP, exhibiting linear relationship in two different concentration ranges: from 2.5 to 40μM and from 62.5 to 117.5μM, with a detection limit of 0.39μM (S/N=3) at an applied potential of −0.55V. The fast current response and good stability were obtained on the HRP modified electrode.

A neuroanatomical correlate of sensorimotor recovery in response to repeated vaginocervical stimulation in rats

Gentle probing against the cervix via the vagina (vaginocervical stimulation, VCS) increases tail flick latency (TFL) to radiant heat; greater force abolishes the tail flick response and other withdrawal responses. This effect occurs in spinal cord-transected rats and in intact rats. On the basis of our earlier finding that VCS releases vasoactive intestinal peptide (VIP) into the spinal cord, and others’ reports of neurotrophic effects of VIP in vitro, we hypothesized that repeated VCS would stimulate sprouting and sensorimotor function of terminals of genital nerve primary afferents in the sacral spinal cord. To test this hypothesis, in the present study, we denervated the genital tract only unilaterally, which significantly reduced the TFL-elevating effect of VCS. Then we applied repeated daily VCS for 1 week and compared the subsequent effectiveness of acute VCS in elevating TFL. The rats that received the repeated daily VCS showed a significantly greater elevation in TFL in response to acute VCS than control rats that did not receive the repeated stimulation. Then, to test whether daily repeated VCS stimulates sprouting of genital primary afferents in such unilaterally genital tract-denervated rats, we transected the contralateral remaining intact pelvic nerve, applied horseradish peroxidase (HRP) to its proximal cut end for 1–2 h, and 2–3 days later counted HRP particles in its terminal zone (L6–S1) in the spinal cord. There were significantly more HRP particles in the rats that received the daily repeated VCS than in the control rats. In the context of these findings, we conclude that VCS in rats can produce a functional sensorimotor recovery via a neurotrophic effect on compromised primary afferents in the spinal cord.

Modulation of endogenous peroxidase by exogenous peroxidase in chinese red radish seedling

To investigate the peroxidase function and its signal pathway, activity and isozyme pattern of peroxidase and transcription level of the peroxidase gene rsprx1 and rsprx2 in Chinese red radish seedling (Raphanus sativus L.) were investigated after administration of the exogenous peroxidase (extract of radish). The activity and isozyme pattern of peroxidases were increased, among cotyledon, hypocotyl and root, the most significant increase of peroxidases activity was observed in hypocotyls, the activity of phenylalanine ammonialyase was also increased, but the pelargonidin content was decreased. Although the exogenous application of horseradish peroxidase and the extract of radish could both affect the activity of endogenous enzyme and metabolites of Chinese red radish seedlings, the latter was more effective than the former. Nifedipine, as L-calcium channel blocker, affected the activity of peroxidase and pelargonidin content in Chinese red radish seedling. Exogenously added nifedipine with the extract caused reversal of the effects. It is concluded that calcium was involved in the signal pathway of the extract of radish in Chinese red radish seedling.

Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.

Interhemisphere Connections of Eye Dominance Columns in the Cat Visual Cortex in Conditions of Impaired Binocular Vision

Data from studies of interhemisphere connections in fields 17 and 18 of cats reared in conditions of impaired binocular vision (monocular deprivation, uni- and bilateral strabismus) are presented. Monosynaptic connections between neurons were studied by microiontophoretic application of horseradish peroxidase into cortical eye dominance columns and the distributions of retrograde labeled callosal cells were analyzed. Spatial asymmetry and eye-specific interhemisphere neuron connections persisted in conditions of monocular deprivation and strabismus. Quantitative changes in connections were less marked in monocular deprivation than strabismus. In cats with impaired binocular vision, as in intact animals, the widths of callosal-receiving zones were greater than the widths of the callosal cell zones, which is evidence for the non-reciprocity of interhemisphere connections in cortical areas distant from the projection of the vertical meridian. Morphofunctional differences between cells mediating connections in the opposite directions are proposed.

Research on the application of horseradish peroxidase and hydrogen peroxide to the oil removal of oily water

Horseradish peroxidase (HRP) has the advantages of low cost, obtaining convenience, broad substrate specificity, etc, so it may be a suitable kind of enzyme to remove dilute emulsified oil and dissolved oil from water. The effect factors of the oil removal using HRP, such as pH, concentrations of HRP and H2O2, additives, Fe2+ ion, temperature and reaction time were researched. When the initial oil concentration was 120 mg/L, the remaining oil concentrations of the synthetic oily water and an actual oily wastewater reached 24.83 mg/L and 21.30 mg/L, respectively. The treatment of oily water using HRP and H2O2 is feasible. It has good application prospects.

Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment

Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H 2O 2 significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H 2O 2 concentrations and incubation time may be needed, depending on nature of sample and should therefore always be individually determined for each study.

Lead poisoning and the blood-brain barrier

Lead exposure may produce varying degrees of neuropsychiatric manifestations from discrete phenomena, quite often seen in children and as an occupational disease, to the rate fulminant lead encephalopathy. It was determined whether or not damage of the blood-brain barrier permeability in adult rats, as has been demonstrated in neonatal animals exposed to lead, could also play a role. Massive lead exposure did not induce any change in the transfer (facilitated diffusion) of phenylalanine any tyrosine measured by means of the indicator dilution technique. Ultrastructural examination, after application of horseradish peroxidase, did not reveal any pathological changes in the permeability to the tracer. It is concluded that in adult rats, in contrast to neonatal animals, the observed pathological signs clearly seen in the chronically exposed animals must be ascribed to a noxious influence of lead on the extravascular side of the blood-brain barrier.

A novel application of horseradish peroxidase: Oxidation of alcohol ethoxylate to alkylether carboxylic acid

A novel application of horseradish peroxidase (HRP) in the oxidation of alcohol ethoxylate to alkylether carboxylic acid in the present of H 2O 2 was reported in this paper. We propose the mechanism for the catalytic oxidation reaction is that the hydrogen transfers from the substrate to the ferryl oxygen to form the α-hydroxy carbon radical intermediate. The reaction offers a new approach for further research structure and catalytic mechanism of HRP and production of alkylether carboxylic acid.

Remodeling of sensory nerve projection onto Mauthner cell during development in zebrafish larva

Although visually evoked Mauthner cell (M-cell) startle responses occur in the goldfish, the afferent projections underlying these reactions have not been previously studied. We have recorded from the M-cell while stimulating the left optic nerve and/or right optic tectum and have traced projections of the optic nerve and restricted areas of the optic tectum using HRP histochemistry and autoradiography. Tectal stimulation elicits similar postsynaptic potentials (PSPs) in both M-cells. The responses recorded in the right (ipsilateral) cell were localized to its ventral dendrite. The existence of uncrossed tectal projections to the ventral dendrite was confirmed morphologically following application of horseradish peroxidase (HRP) to the optic tectum. The PSPs contained both inhibitory and excitatory components, but with adequate stimulus strength, excitation of either M-cell dominated. Thus, this pathway is probably sufficient to trigger visually evoked startle responses mediated by the M-cell. Stimulation of the left optic nerve also evoked PSPs capable of bringing both M-cells to threshold. The blockage of this response by conditioning stimulation of the right tectum suggests that the visual information is relayed to the M-cells through this structure. In support of these findings, no label was found near any portion of the M-cell after either intraocular injection of tritiated proline or application of HRP to the cut end of the optic nerve. In summary, visual input to the M-cell is mediated via projections from the tectum, is segregated onto the ventral dendrite, and is capable of bringing this neuron to threshold. This pathway presumably accounts for the demonstrated behavioral efficacy of visual stimuli in evoking a startle response.

Interhemisphere connections of the visual cortex in cats with bilateral strabismus

The distribution of retrograde labeled callosal cells after microiontophoretic application of horseradish peroxidase into individual cortical columns in fields 17 and 18 was studied in cats reared with bilateral strabismus (with an angle of eye deviation of 10-35 degrees ). The area containing labeled cells was located asymmetrically in relation to the position of the injected column in the opposite hemisphere. Some of the cells were located in those parts of the transitional zone between fields 17 and 18 whose retinotopic coordinates corresponded to the column coordinates (as in intact cats). Other labeled cells were located in fields 17 and 18 and were grouped into clusters located at distances of about 1000 microm from the marginal clusters of the transitional zone. The locations of labeled cells in the lateral geniculate body showed that most columns receive inputs from the ipsilateral eye. Evidence for eye specificity of these monosynaptic interhemisphere connections is presented. The functional significance of changes in these connections in bilateral strabismus is discussed.

NK1 receptor activation by geniohyoid primary afferents modulates parasympathetic postganglionic neuronal excitability in the rat

Previous studies have indicated that the geniohyoid (GH) muscle receives innervation via both the hypoglossal nerve (CNXII) and the ansa cervicalis. Our recent studies revealed that the efferent root that contributes to the ansa cervicalis is a parasympathetic pathway and contains postganglionic cell bodies. Afferent axons from the GH muscle also travel via the ansa cervicalis, and afferent cell bodies are located in spinal ganglia. The present study attempts to locate the central terminations of these afferents. From the peripheral cut end of the ansa cervicalis, we recorded afferent discharges that coincided with inspiration and these were elicited by stretch of the GH muscle. After cutting CNXII proximal to its union with the ansa cervicalis, we applied horseradish peroxidase to the branch of CNXII that innervates the GH muscle. This procedure labeled cells ipsilaterally in the C2 spinal ganglia but not in the brainstem or upper spinal cord. Substance P-reactive terminals in the peripheral CNXII trunk were in apparent contact with vasoactive intestinal peptide-reactive cell bodies. Addition of the NK1 receptor agonist SP NK1 excited parasympathetic postganglionic neurons and the specific NK1 receptor antagonist GR82334 blocked these effects in vitro. These results suggest that GH primary afferents synapse on parasympathetic postganglionic neurons in the CNXII trunk and that activation of SP NK1 receptors modulates activity in these neurons.