Encapsulated Horseradish Peroxidase Research Articles

Magnetic iron oxide nanoparticles encapsulating horseradish peroxidase (HRP): synthesis, characterization and carrier for the generation of free radicals for potential applications in cancer therapy

We report synthesis of iron oxide nanoparticles encapsulating HRP. The average diameter of the particles was around 20 nm. HRP has been used to convert IAA to a toxic oxidized product and its toxic effect has been seen on cancerous cell lines.

Liposomal delivery of horseradish peroxidase for thermally triggered injectable hyaluronic acid-tyramine hydrogel scaffolds.

A thermoresponsive injectable hydrogel scaffold for tissue engineering has been developed, whereby the scaffold was injected as a liquid at room temperature, and gelled at the target site in response to the change in body temperature. Our approach involved suspending thermoresponsive liposomes, which encapsulated horseradish peroxidase (HRP), in a hyaluronic acid-tyramine (HA-Tyr) conjugate and hydrogen peroxide (H2O2) solution. At room temperature, HRP was separated from the HA-Tyr conjugate by the lipid membrane, and hence the precursor solution remained as a liquid and was injectable. Upon injection and exposure to body temperature, the lipids experienced a phase transition, which significantly increased the membrane permeability and led to the release of HRP and the oxidative coupling of Tyr moieties with H2O2, forming a crosslinked hydrogel scaffold. It was shown in this study that the precursor solution remained as a liquid on the order of hours at 20 °C, yet gelation could be induced within minutes by heating to 37 °C. Furthermore, it was shown that HRP release could be controlled by various material and processing parameters, and that the gelation rate could be adjusted to meet various clinical needs by adjusting HRP encapsulation, liposome concentration and HA-Tyr concentration.

Organophosphate vapor detection on gold electrodes using peptide nanotubes

Peptide nanotubes (PNTs) encapsulating horseradish peroxidase and surface coated with acetylcholinesterase (AChE) were attached to gold screen printed electrodes to construct a novel gas phase organophosphate (OP) biosensor. When the sensor with the AChE enzyme is put in contact with acetylthiocholine (ATCh), the ATCh is hydrolyzed to produce thiocholine, which is then oxidized by horseradish peroxidase (HRP). Direct electron transfer between HRP and electrode is achieved through PNTs. The signal produced by the electron transfer is measured with cyclic voltammetry (CV). The presence of an OP compound inhibits this signal by binding with the AChE enzyme. In this study, gas phase malathion was used as a model OP due to the fact that it displays the identical binding mechanism with acetylcholinesterase (AChE) as its more potent counterparts such as sarin and VX, but has low toxicity, making it more practical and safer to handle. The CV signal was proportionally inhibited by malathion vapor concentrations as low as 12ppbv. Depending on the method used in their preparation, the electrodes maintained their activity for up to 45 days. This research demonstrates the potential of applying nano-modified biosensors for the detection of low levels of OP vapor, an important development in countering weaponized organophosphate nerve agents and detecting commercially-used OP pesticides.

Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal

In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2-chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization.

Photoreaction of a Hydroxyalkyphenone with the Membrane of Polymersomes: A Versatile Method To Generate Semipermeable Nanoreactors

Block copolymer vesicles can be turned into nanoreactors when a catalyst is encapsulated in these hollow nanostructures. However the membranes of these polymersomes are most often impermeable to small organic molecules, while applications as nanoreactor, as artificial organelles, or as drug-delivery devices require an exchange of substances between the outside and the inside of polymersomes. Here, a simple and versatile method is presented to render polymersomes semipermeable. It does not require complex membrane proteins or pose requirements on the chemical nature of the polymers. Vesicles made from three different amphiphilic block copolymers (α,ω-hydroxy-end-capped poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA), α,ω-acrylate-end-capped PMOXA-b-PDMS-b-PMOXA, and poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PB)) were reacted with externally added 2-hydroxy-4'-2-(hydroxyethoxy)-2-methylpropiophenone under UV-irradiation. The photoreactive compound incorporated into the block copolymer membranes independently of their chemical nature or the presence of double bonds. This treatment of polymersomes resulted in substantial increase in permeability for organic compounds while not disturbing the size and the shape of the vesicles. Permeability was assessed by encapsulating horseradish peroxidase into vesicles and measuring the accessibility of substrates to the enzyme. The permeability of photoreacted polymersomes for ABTS, AEC, pyrogallol, and TMB was determined to be between 1.9 and 38.2 nm s(-1). It correlated with the hydrophobicity of the compounds. Moreover, fluorescent dyes were released at higher rates from permeabilized polymersomes compared to unmodified ones. The permeabilized nanoreactors retained their ability to protect encapsulated biocatalysts from degradation by proteases.

Enzyme activity assay for horseradish peroxidase encapsulated in peptide nanotubes

Encapsulation of horseradish peroxidase (HRP) inside a peptide nanotube (PNT) was demonstrated and its activity was measured. Enzyme assay verified that 0.16μg of the enzymes were encapsulated in 1mg of PNTs. The encapsulation was also verified with TEM, UV–vis spectroscopy, and FTIR. The activity of the encapsulated HRP was examined for thermal stability, long-term storage stability, and resistance to a denaturant. They showed good storage stability, retaining its activity up to 90%, while the free HRP lost 50% of its activity over the course of 18days. At 55°C, the encapsulated HRP activity remained 20% higher than that of the free HRP. With the denaturant, guanidinium hydrochloride (GdmHCl), the encapsulated HRP activity was maintained around 10% higher than the free HRP. This result proves that the encapsulation of HRP inside the PNT may be an effective way to keep the enzyme activity stable in various environments.

Core/Shell Nanofibers with Embedded Liposomes as a Drug Delivery System

The broader application of liposomes in regenerative medicine is hampered by their short half-life and inefficient retention at the site of application. These disadvantages could be significantly reduced by their combination with nanofibers. We produced 2 different nanofiber-liposome systems in the present study, that is, liposomes blended within nanofibers and core/shell nanofibers with embedded liposomes. Herein, we demonstrate that blend electrospinning does not conserve intact liposomes. In contrast, coaxial electrospinning enables the incorporation of liposomes into nanofibers. We report polyvinyl alcohol-core/poly-ε-caprolactone-shell nanofibers with embedded liposomes and show that they preserve the enzymatic activity of encapsulated horseradish peroxidase. The potential of this system was also demonstrated by the enhancement of mesenchymal stem cell proliferation. In conclusion, intact liposomes incorporated into nanofibers by coaxial electrospinning are very promising as a drug delivery system.

Mixed monolayers of ferrocenylalkanethiol and encapsulated horseradish peroxidase for sensitive and durable electrochemical detection of hydrogen peroxide.

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H(2)O(2) at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC(11)SH) mediator, facile electron transfer between FcC(11)SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H(2)O(2) reduction (to H(2)O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H(2)O(2) (to O(2)). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H(2)O(2) to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (>3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC(11)SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H(2)O(2) present in complex sample media.

Removal of Phenols with Encapsulated Horseradish Peroxidase in Calcium Alginate

Horseradish peroxidase was encapsulated in calcium alginate for the purpose of phenol removal. Considering enzyme encapsulation efficiency, retention activity and enzyme leakage of the capsules, the best gelation condition was found to be 1 % w/v of sodium alginate solution and 5.5 % w/v of calcium chloride hexahydrate. Upon immobilization, pH profile of enzyme activity changes as it shows higher value at basic and acidic solution. Besides, for each phenol concentration there would be an enzyme concentration which going further than this value has no significant effect on phenol removal. Investigation into time course of phenol removal for both encapsulated and free enzyme showed that encapsulated enzyme had nearly similar efficiency in comparison with the same concentration of free enzyme; however the capsules were reusable up to four cycles without any changes in their efficiency. The ratio of hydrogen peroxide/phenol at which highest phenol removal obtained, found to be dependent on initial phenol concentration and in the solution of 2 and 8 mM phenol it were 1.15 and 0.94 respectively.

Horseradish peroxidase microcapsules based on layer-by-layer polyelectrolyte deposition

Polyelectrolyte multilayer microcapsules have been prepared for encapsulating horseradish peroxidase (HRP). The CaCO 3 microparticles containing HRP molecules were prepared and subsequently enwrapped with layer-by-layer (LbL) deposited multilayer films. The LbL films were constructed via an alternate electrostatic adsorption of poly(allylamine) and poly(styrene sulfonate). The CaCO 3 templates were then dissolved in an ethylenediaminetetraacetic acid solution, leaving HRP in the microcapsules. Optical microscope and fluorescence microscope observations indicated that the microcapsules are homogeneous spheres with an average diameter about 7 μm. The encapsulated HRP showed catalytic activity in the oxidation of pyrogallol, suggesting the microcapsules provide a suitable environment for enzymatic reaction. A leakage of HRP from the microcapsules was negligibly small. Therefore, the HRP-containing microcapsules is promising for detection and treatment of phenolic compounds.

Amperometric H2O2 biosensor based on poly-thionine nanowire/HRP/nano-Au-modified glassy carbon electrode

Poly-thionine nanowire/HRP/nano-Au composite nanomaterial was developed to fabricate the H2O2 biosensor. Poly-thionine nanowires (PTHNWs) have been synthesized by electrodeposition in porous anodic aluminum oxide (AAO) template. The morphological characterization of PTHNWs was examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). The nanowires prepared by this method can be used to encapsulate horseradish peroxidase (HRP) and nano-Au by in situ electrochemical copolymerization. The resulting PTHNWs–HRP–nano-Au material facilitates electron-transfer process in electrochemical sensor design. The PTHNWs–HRP–nano-Au film-modified electrode showed to be excellent amperometric sensors for H2O2. In pH 6.98 phosphate buffer, almost interference-free determination of H2O2 is realized at −0.1 V versus SCE with a linear range of 5 × 10−7 to 1.3 × 10−2 M, a correlation coefficient of 0.998 and response time <5 s. The sensitivity of the H2O2 biosensor is up to 168 μA mM−1 cm−2 and the detection limit is 0.3 μM. Furthermore, the biosensor exhibited long-term stability, and good reproducibility.

Application of SiO 2–poly(dimethylsiloxane) hybrid material in the fabrication of amperometric biosensor

Silica xerogel was widely used in the development of biosensors by coupling the desired biological components to various transducers. Unfortunately, the application of xerogels is limited due to their poor mechanical properties and poor structural maintenance of entrapped biomaterials. In this study, the availability of poly(dimethylsiloxane) (PDMS)-modified silica sol–gel (TEOS/PDMS Ormosil) glass in the immobilization of enzymes and its application in the fabrication of amperometric biosensor was investigated. We demonstrated that most of activity of encapsulated horseradish peroxidase can be preserved in PDMS-modified SiO 2 glass compared with conventional silica xerogel. The synthesized monoliths exhibited high transparency and crack-free. The enzyme electrode based on glucose oxidase encapsulated TEOS/PDMS Ormosil glass on Pd electrode was fabricated and characterized electrochemically. The characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The biosensor exhibited a series of good properties: high sensitivity (1.63 μA mM −1 analyte), short response time and high reproducibility. The results indicate that TEOS/PDMS Ormosil glass has great potential to the immobilization of biomaterials as well as the fabrication of biosensors.

Ultraviolet Photolithographic Development of Polyphosphazene Hydrogel Microstructures for Potential Use in Microarray Biosensors

Polyphosphazenes that bear both methoxyethoxyethoxy and cinnamyl side groups were synthesized and evaluated for use as hydrogels incorporated into micrometer-scale biosensor arrays. Polymers with the general formula [NPRxR‘y]n where R = OCH2CH2OCH2CH2OCH3 and R‘ = OCHCHCH2Ph (x = y = 1; x = 2, y = 0) were synthesized. The polymers were cross-linked to form hydrogels by exposure to ultraviolet radiation (λ = 320−480 nm) in the presence of a photoinitiator. Hydrogel microstructures in the size range 50−500 μm were fabricated using standard photolithographic techniques. The resolution and dimensions of these microstructures were examined by optical microscopy, scanning electron microscopy, and profilometry. The resultant three-dimensional hydrogel microstructures were used to encapsulate enzymes for biosensor applications. The enzymatic activity of encapsulated horseradish peroxidase (HRP) was examined as a model system. The HRP catalyzed reaction between H2O2 and Amplex Red to produce a fluorescent product,...

Fluorescein chemiluminescence method for estimation of membrane permeability of liposomes

Unilamellar liposomes have a compartment enclosed by a single lipid bilayer. Encapsulation of compounds, such as drugs, genes, and enzymes, into liposomes has been applied successfully to drug delivery systems and cosmetics [1,2]. For these applications of liposomes, control, and characterization of release and leakage of compounds are very important. Release and leakage are attributable to the membrane permeability, and in some cases to instability, of the liposomes. The assay of membrane permeability is generally performed by monitoring the release of fluorescein derivatives, such as 5(6)-carboxyfluorescein and calcein (bis[N,N 0-di(carboxymethyl)-aminoethyl] fluorescein), encapsulated in liposomes [3,4]. In these assays, the dyes are encapsulated in liposomes at concentrations greater than 50–100 mM, where more than 95% of fluorescence of the dyes is self-quenched. Release of the dyes inside liposomes leads to an increase in fluorescence intensity due to the reduction of self-quenching. The percentage of released dye over a certain period is used as an index of membrane permeability. The dyes exhibit low permeability to bilayer because the dyes have multiple negative charges. Release of the dyes, therefore, requires several hours or days. It is generally difficult to use fluorescein for dye-release assay because encapsulated fluorescein leaks rapidly from the inner phase of liposomes during separation of the free dye. On the other hand, we previously reported a chemiluminescence (CL) reaction inside liposomes encapsulating horseradish peroxidase

Inorganic–Organic Hybrid Nanoparticles from n-Octyl Triethoxy Silane

Ormosil (organically modified silane) such as n-octyl triethoxy silane has been found to aggregate in the form of normal micelles as well as reverse micelles in which the triethoxy silane moeities are hydrolyzed to form a hydrated silica network while the n-octyl groups are held together through hydrophobic interaction. These nanoparticles are spherical in shape and are nearly monodispersed with an average diameter of below 100 nm. The nanoparticles originating from the micellar aggregate have an hydrophobic core with a layer of the hydrated silica network at the surface. The hydrophobic core can host hydrophobic molecules such as tetraphenyl porphyrin, which is leached out of the particles extremely slowly compared to that in Triton X-100 micelles. The nanoparticles originating from the reverse micelles have a hydrated silica network in the core surrounded by the hydrophobic n-octyl chains on the particle surface. The hydrophilic silica cores of these nanoparticles have been used to encapsulate horseradish peroxidase (HRP) and the enzyme shows its activity and follows Michaelis–Menten kinetics.

Sol-gel encapsulated horseradish peroxidase: a catalytic material for peroxidation.

This study addresses the viability of sol-gel encapsulated HRP (HRP:sol-gel) as a recyclable solid-state catalytic material. Ferric, ferric-CN, ferrous, and ferrous-CO forms of HRP:sol-gel were investigated by resonance Raman and UV-visible methods. Electronic and vibrational spectroscopic changes associated with changes in spin state, oxidation state, and ligation of the heme in HRP:sol-gel were shown to correlate with those of HRP in solution, showing that the heme remains a viable ligand-binding complex. Furthermore, the high-valent HRP:sol-gel intermediates, compound I and compound II, were generated and identified by time-resolved UV-visible spectroscopy. Catalytic activity of the HRP:sol-gel material was demonstrated by enzymatic assays by using I(-), guaiacol, and ABTS as substrates. Encapsulated HRP was shown to be homogeneously distributed throughout the sol-gel host. Differences in turnover rates between guaiacol and I(-) implicate mass transport of substrate through the silicate matrix as a defining parameter in the peroxidase activity of HRP:sol-gel. HRP:sol-gel was reused as a peroxidation catalyst for multiple reaction cycles without loss of activity, indicating that such materials show promise as reusable catalytic materials.