Adsorption Of Horseradish Peroxidase Research Articles

Stopped Flow of Glycerol Induces the Enhancement of Adsorption and Aggregation of HRP on Mica.

Glycerol is a usable component of heat-transfer fluids, and is thus suitable for the use in microchannel-based heat exchangers in biosensors and microelectronic devices. The flow of a fluid can lead to the generation of electromagnetic fields, which can affect enzymes. Herein, by means of atomic force microscopy (AFM) and spectrophotometry, a long-term effect of stopped flow of glycerol through a coiled heat exchanger on horseradish peroxidase (HRP) has been revealed. Samples of buffered HRP solution were incubated near either the inlet or the outlet sections of the heat exchanger after stopping the flow. It has been found that both the enzyme aggregation state and the number of mica-adsorbed HRP particles increase after such an incubation for 40 min. Moreover, the enzymatic activity of the enzyme incubated near the inlet section has been found to increase in comparison with that of the control sample, while the activity of the enzyme incubated near the outlet section remained unaffected. Our results can find application in the development of biosensors and bioreactors, in which flow-based heat exchangers are employed.

Development of Targeted Protein-Displaying Technology with a Novel Carbon Material

This study reports a new carbon material and its specific display of targeted protein. The properties of the carbon materials fabricated with carbon black MOGUL® were analyzed. The carbon materials were spherical structures with 55.421 µm as a median value. The specific surface area, pore volume, average pore diameter, and total of the acidic functional group were 130 m2·g-1, 0.55 cm3·g-1, 17.2 nm, and 0.29 mEq·g-1, respectively. The adsorption-desorption isoform of the carbon materials showed type IV of the hysteresis loop as defined by IUPAC, indicating non-uniform mesoporous structures (2-50 nm). The distribution of the log differential pore volume also indicated non-uniform porous structures because (i) the difference between the average pore size and the most frequent pore size was significant and (ii) the σ value was larger than the average value regarding the pore sizes. However, 10-90% of the integrated values of the log differential pore volume were 57.4% of the total integrated values, and the distribution was similar to the Gauss distribution model. Although the value of the total of the acidic functional group was 2.5-5.4 times lower than the values of the HPLC columns, the carbon materials require good scaffold quality rather than good HPLC quality. Therefore, the amounts could be enough for the scaffold of biotin hydrazide. To demonstrate the property of displaying the targeted proteins, carbon materials displaying biotin hydrazide by covalent bonding were prepared and avidin-labeled horse radish peroxidase (HRP) was bound to the biotin region. The carbon materials were porous structures, so the unspecific adsorption of HRP was estimated. Then, the maintenance ratios of HRP activities were analyzed in the repeated-use-with-wash processes after each evaluation, resulting in the activities of HRP on the carbon materials being treated with biotin hydrazide being significantly maintained compared to that of the ones without biotin hydrazide. The study revealed the properties of the carbon materials and indicated the display of HRP, suggesting that the carbon materials could be a new material for displaying targeted proteins.

Effect of a Conical Cellulose Structure on Horseradish Peroxidase Biomacromolecules

The effect of a dielectric conical structure on the adsorption properties of an enzyme on mica was studied by atomic force microscopy (AFM) with the example of horseradish peroxidase (HRP). The cone used was a cellulose cone with a 60° apex angle. Namely, AFM allowed us to reveal an increase in the enzyme’s aggregation during its adsorption onto mica from the solution incubated near the cone apex for 40 min—as compared with the control enzyme samples incubated far away from the cone. In contrast, no change in the HRP adsorption properties was observed after shorter (10 min) incubation of the sample near the cone. The enzymatic activity of HRP was found to be the same for all the enzyme samples studied. Our findings should be considered upon designing biosensors (in particular, those intended for highly sensitive diagnostic applications) and bioreactors containing conical structural elements. Furthermore, since HRP is widely employed as a model enzyme in studies of external impacts on enzymes determining food quality, our data can be of use in the development of food-processing methods based on the use of electromagnetic radiation (microwave treatment, radiofrequency heating, etc.).

The Effect of a Dodecahedron-Shaped Structure on the Properties of an Enzyme

In this research, the influence of a dodecahedron-shaped structure on the adsorption behavior of a horseradish peroxidase (HRP) enzyme glycoprotein onto mica substrates was studied. In the experiments, samples of an aqueous HRP solution were incubated at various distances (0.03 m, 2 m, 5 m, and control at 20 m) from the dodecahedron surface. After the incubation, the direct adsorption of HRP onto mica substrates immersed in the solutions was performed, and the mica-adsorbed HRP particles were visualized via atomic force microscopy (AFM). The effect of the increased HRP aggregation was only observed after the incubation of the enzyme solution at the 2 m distance from the dodecahedron. In addition, with respect to the control sample, spectrophotometric measurements revealed no change in the HRP enzymatic activity after the incubation at any of the distances studied. The results reported herein can be of use in the modeling of the possible influences of various spatial structures on biological objects in the development of biosensors and other electronic equipment.

Improvement of enzymatic properties and decolorization of azo dye: immobilization of horseradish peroxidase on cationic maize starch

The main objective of this work was designed to immobilize the horseradish peroxidase (HRP) into an efficient modified natural polymers carrying positive charges. Thus, cationic maize starch (CMS) was chemically synthesized thru the etherifying agent for native maize starch using sodium hydroxide as a base to deprotonate the hydroxyl groups in the starch and (S)-1-amino-3-chloropropan-2-ol (CHPA) as etherifying reagent. CMS was immobilized with horseradish peroxidase (HRP) to improve its characteristics under a variety of conditions. The findings demonstrated that, at 150 mg CMS, 250 units of HRP, and pH 7.0, the highest immobilization effectiveness of HRP (91%) was achieved. The morphological surface structure depicted via field emission-scanning electron microscope (FE-SEM) and roughness assessments proved that the surface pores and roughness were completely blocked and became rough owing to the adsorption of HRP onto the surface of CMS. The immobilized enzyme was reused 10 times, with 65% of its activity remaining. For immobilized HRP on CMS, the pH optimum of soluble HRP was adjusted from broad (6.0–7.0) to sharp (7.0). The thermal stability of the immobilized enzyme was greater improved while immobilized into CMS. The results illustrated that some of the phenolic compounds were oxidized more efficiently by the immobilized enzyme. Heavy metals and denatured compounds had no impact on the immobilized enzyme. After 1 h of incubation, the immobilized HRP decolorized 97% of the utilized methyl orange (MO) as azo dye model, whereas the soluble form removed 72% of dye. Thus, our findings suggested that the cationized starch immobilized with enzyme could be considered as an alternative effective material for the removal of colored industrial wastewaters. • Cationic maize starch (CMS) was prepared using 3-chloro 2-hydroxy propyl amine (CHPA). • The immobilized HRP reused 10 times with retention 65% of its activity. • The immobilized enzyme resisted the effect of heavy metals and denatured compounds.

Adsorption of horseradish peroxidase on metallic nanoparticles -effects on reactive oxygen species detection using 2'-7'dichlorofluorescin diacetate

Adsorption of horseradish peroxidase on metallic nanoparticles -effects on reactive oxygen species detection using 2'-7'dichlorofluorescin diacetate

AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure

In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid’s apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.

Adsorption of Horseradish Peroxidase on Metallic Nanoparticles: Effects on Reactive Oxygen Species Detection Using 2',7'-Dichlorofluorescin Diacetate.

The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study acellular reactive oxygen species (ROS) production from nanoparticles (NPs). This study examined whether HRP adsorbs onto NPs of Mn, Ni, and Cu and if this surface process influences the extent of metal release and hence the ROS production measurements using the DCFH assay in phosphate buffered saline (PBS), saline, or Dulbecco’s modified Eagle’s medium (DMEM). Adsorption of HRP was evident onto all NPs and conditions, except for Mn NPs in PBS. The presence of HRP resulted in an increased release of copper from the Cu NPs in PBS and reduced levels of nickel from the Ni NPs in saline. Both metal ions in solution and the adsorption of HRP onto the NPs can change the activity of HRP and thus influence the ROS results. The effect of HRP on the NP reactivity was shown to be solution chemistry dependent. Most notable was the evident affinity/adsorption of phosphate toward the metal NPs, followed by a reduced adsorption of HRP, the concomitant reduction in released manganese from the Mn NPs, and increased levels of released metals from the Cu NPs in PBS. Minor effects were observed for the Ni NPs. The solution pH should be monitored since the release of metals can change the solution pH and the activity of HRP is known to be pH-dependent. It is furthermore essential that solution pH adjustments are made following the addition of NaOH during diacetyl removal of DCFH-DA. Even though not observed for the given exposure conditions of this study, released metal ions could possibly induce agglomeration or partial denaturation of HRP, which in turn could result in steric hindrance for H2O2 to reach the active site of HRP. This study further emphasizes the influence of HRP on the background kinetics, its solution dependence, and effects on measured ROS signals. Different ways of correcting for the background are highlighted, as this can result in different interpretations of generated results. The results show that adsorption of HRP onto the metal NPs influenced the extent of metal release and may, depending on the investigated system, result in either under- or overestimated ROS signals if used together with the DCFH assay. HRP should hence be used with caution when measuring ROS in the presence of reactive metallic NPs.

Poly(amino acid) Multilayers Modified Dendritic Mesoporous Silica Nanoparticles Achieve Effective Enzyme Stability for Ultrasensitive Immunoassay.

Enzyme-linked immunosorbent assay (ELISA) is one of the most common techniques in biomedical detection; however, the poor sensitivity in early diagnosis for some diseases seriously limits its application. In this work, we developed an ultrasensitive ELISA system that is based on horseradish peroxidase (HRP)-loaded dendritic mesoporous silica nanoparticles (DMSN) modified with poly(amino acid) multilayers (defined as DSHP). A large amount of HRP adsorption was achieved in center-radial mesoporous channels of DMSN because of the high specific surface area and large pore size, leading to significant signal amplification. Additionally, DSHP could not only effectively maintain HRP activity for at least 10 days but also provide preferable protection for HRP activity even at high temperatures or a wide pH range. Moreover, the DSHP system exhibited admirable signal amplification performance with a limit of detection of 0.667 fM and a wide detectable range from 6.67 × 10-4 to 6.67 × 105 pM, whose sensitivity was 104 times higher than that of the conventional ELISA. We believe that the DSHP will offer a new strategy for signal amplification of the ELISA system in clinical diagnosis.

Immobilization of horseradish peroxidase onto Purolite® A109 and its anthraquinone dye biodegradation and detoxification potential.

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite® A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 ± 1.41 U g-1 and 85.71 ± 1.62 U g-1 after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (~90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.

Carbon-based electrode loaded with Y-doped SrTiO3 perovskite as support for enzyme immobilization in biosensors

Many studies have been presented Y-doped SrTiO3 as a potential alternative material for electrical, electrochemical and chemical applications due to its mixed (ionic and electronic) conduction and its morphological characteristics. In this work, 8% mol yttrium doped SrTiO3 (YST08) was used to assemble a Horseradish peroxidase (HRP) biosensor aiming at hydrogen peroxide (H2O2) detection. The perovskite was characterized by X-ray diffraction (XRD), Brunauer–Emmett–Teller N2 physisorption (BET), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS); and, after HRP immobilization, by zeta potential, atomic force microscopy (AFM), cyclic voltammetry, Fourier-transform infrared spectroscopy (FTIR), and chronoamperometry. The structure presented a low specific surface area due to particle agglomeration as a result of the elevated temperature of calcination. This characteristic was ratified by SEM analysis, which also demonstrated regular and well-rounded grains. Zeta potential results suggest successful HRP immobilization on YST08 surface because of the resemblance of zeta potential of immobilized sample and free HRP. FTIR spectra and AFM micrographs confirm a homogenous HRP adsorption due to functional groups identification and surface topography changes. YST08 + HRP electrode proved to be efficient in sensing H2O2 in different scan rates and background electrolytes under electrochemical evaluation. Results also showed that the process is surface-controlled, exhibiting a low reduction potential for H2O2 around −0.31 V at pH = 7. The YST08 matrix was effective to the diffusion mechanisms presenting a detection limit of 14.97 μM; and increases the enzyme long-term activity, retaining 14.97% of specific capacity after 180 days.

A close-packed imprinted colloidal array for naked-eye detection of glycoproteins under physiological pH

According to the combination of colloidal crystals and molecular imprinting techniques, a novel close-packed imprinted colloidal array (CPICA) for naked-eye horseradish peroxidase (HRP) detection at physiological pH was proposed. The CPICA was fabricated by self-assemble of monodispersed HRP imprinted particles. The HRP imprinted particles were prepared based on surface imprinting technique and immobilized template strategy using 2,4-difluoro-3-formylphenylboronic acid (DFFPBA) as functional monomer which allowed the material binding of HRP at physiological pH (denoted as SiO2@DFFPBA/MIPs). The adsorption capacity of the SiO2@DFFPBA/MIPs for HRP was 1.16 μmol/g, and reached saturated adsorption within 25 min. The limit of detection (LOD) of the CPICA was 3.0 × 10−13 mol/mL. In addition, the adsorption of HRP on the CPICA could be directly transferred into visible color changes and readable optical signals through the reflection peak shifts. The structure color of the CPICA changed from brilliant blue to dark red with an maximum red shift of 87 nm when the HRP concentration increased from 2.5 to 20.0 μmol/L. Moreover, the CPICA could be used to detect HRP from human serum sample, which demonstrated the promising application prospects in colorimetric sensors.

Control of Adsorption Horseradish Peroxidase on the Surface of Glass Multicapillary by Using a Polyelectrolyte on Layer-by-Layer Technology

This work aims to explore the effect of polyelectrolyte layers on the protein adsorption using the example horseradish peroxidase (HRP) in a glass multicapillary to develop ways to control the selective sorption. It is shown that a more effective adsorption of HRP was observed for the bilayer polyethylene (PEI)/polystyrene sulfonate (PSS). This effect is explained by the fact that the polycation PEI modifies the glass surface, and the polyanion PSS creates a layer for the HRP effective sorption.

Electrochemical detection of carcinoembryonic antigen based on silver nanocluster/horseradish peroxidase nanocomposite as signal probe

A new and novel signal probe based on silver nanoclusters (Ag NCs)/horseradish peroxidase (HRP) nanocomposite (Ag NCs-HRP) was developed. The probe was utilized for electrochemical immunoassay for the detection of carcinoembryonic antigen (CEA). The Ag NCs was synthesized via chemical reduction in the presence of DNA strand containing CEA aptamer (ATACCAGCTTATTCAATT) and the Ag NCs synthesis template (C12). After the adsorption of HRP onto Ag NCs surface, the resulted Ag NCs-HRP could bind with CEA and acted as signaling probe. The assay involves the following steps: (1) construction of an immunosensor by immobilizing the antibody against CEA on a glassy carbon electrode; (2) incubate the immunosensor with sample containing CEA; (3) incubate the immunosensor with Ag NCs-HRP; (4) signal amplification via HRP catalyzed deposition of an insulating polymer film, poly(3,3′-dimethoxybenzidine) (PDB) onto electrode surface; (5) measurement via square wave voltammetry in Fe(CN)63−/4− solution. The current decreased due to the formation of insulating PDB film is logarithmically related to the concentration of CEA in the 1pg to 10ngmL−1 range, with a detection limit of 0.5pgmL−1. The developed nanocluster/enzyme signal probe can certainly be modified for the detection of different other trace targets in bioanalytical fields.

An adhesive conducting electrode material based on commercial mesoporous titanium dioxide as a support for Horseradish peroxidase for bioelectrochemical applications

An adhesive conducting electrode material containing of graphite, biocompatible ion exchange polymer nafion® and commercial mesoporous TiO2 impregnated with horseradish peroxidase (HRP) is prepared and characterized by amperometric, UV–vis and N2 sorption methods. The factors influencing the performance of the resulting biosensor are studied in detail. The optimal electrode material consists of 45% graphite, 50% impregnated HRP–TiO2 and 5% nafion®. The optimum conditions for H2O2 reduction are an applied potential of –0.3V and 0.1mM hydroquinone. Sensitivity and limit of detection in the optimum conditions are 1AM−1 cm−2 and 1µM correspondingly. The N2 sorption results show that the pore volume of TiO2 decreases sharply upon adsorption of HRP. The preparation process of the proposed enzyme electrode is straightforward and potentially can be used for preparation of carbon paste electrodes for bioelectrochemical detections.

Photoelectrochemical Detection of H2O2 Based on Flower‐Like CuInS2‐Graphene Hybrid

AbstractA novel photoelectrochemcial biosensing system was fabricated based on the composition of horseradish peroxidase (HRP), flower‐like CuInS2 (CIS) and graphene on indium tin oxide (ITO) electrode for detecting H2O2. The graphene layer was used as highly conductive scaffolds for electron transport from the ITO electrode to CIS. Furthermore, the flower‐like CIS enhanced the multi‐reflection of light and provided matrixes for the adsorption of HRP. Utilizing one‐pot solvothermal method, we prepared flower‐like CIS‐graphene hybrid (GCIS). Electrochemical tests displayed advantage of graphene with better electron conductivity, and HRP/GCIS showed higher photoelectrochemical behavior.

Enzyme modified peptide nanowire as label for the fabrication of electrochemical immunosensor

Ferrocene (Fc) functionalized self-assembled peptide nanowire (Fc-PNW) was synthesized, functionalized and used as label for the fabrication of electrochemical immunosensors to detect substance P (SP). To prepare the label, gold nanoparticles (AuNPs) were immobilized onto the Fc-PNW for the following adsorption of horseradish peroxidase (HRP) and secondary anti-SP antibody (Ab2, Fc-PNW-HRP-Ab2). The role of Fc-PNW in the electrochemical label was not only as supporting matrix for HRP and Ab2, but also as the mediator for HRP to enhance the sensitivity of HRP toward H2O2 detection. The immunosensor was fabricated based on the traditional sandwich protocol with primary anti-SP antibody (Ab1) immobilized onto graphene surface. The catalytic current of HRP toward H2O2 in the presence of mediator Fc was used as signal of the immunosensor. Different parameters, such as the weight ratio of HRP to Ab2 on the Fc-PNW and the antibody–antigen incubation time were optimized to improve the performance of the immunosensor. The resulting immunosensor has high sensitivity, wide linear range (0.01–50ng/mL) and low detection limit (5pg/mL).

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H2O2) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM−1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

Core–shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications

Ag@C core–shell nano-composites have been prepared by a simple one-step hydrothermal method and are further explored for protein immobilization and bio-sensing. The electrochemical behavior of immobilized horseradish peroxidase (HRP) on Ag@C modified indium–tin–oxide (ITO) electrode and its application as H2O2 sensor are investigated. Electrochemical and UV–vis spectroscopic measurements demonstrated that Ag@C nano-composites provide excellent matrixes for the adsorption of HRP and the entrapped HRP retains its bioactivities. It is found that on the HRP–Ag@C/ITO electrode, HRP exhibited a fast electron transfer process and good electrocatalytic reduction toward H2O2. Under optimum experimental conditions the biosensor linearly responds to H2O2 concentration in the range of 5.0×10−7–1.4×10−4M with a detection limit of 2.0×10−7M (S/N=3). The apparent Michaelis–Menten constant (KappM) of the biosensor is calculated to be 3.75×10−5M, suggesting high enzymatic activity and affinity toward H2O2. In addition, the HRP–Ag@C/ITO bio-electrode shows good reproducibility and long-term stability. Thus, the core–shell structured Ag@C is an attractive material for application in the fabrication of biosensors due to its direct electrochemistry and functionalized surface for efficient immobilization of bio-molecules.

Characterization of horseradish peroxidase immobilized on PEGylated polyurethane nanoparticles and its application for dopamine detection

In this work, we present a study of peroxidase adsorption on PEGylated polyurethane (PU-PEG) nanoparticles using a purified horseradish peroxidase (HRP). The influence of the immobilization on the catalytic activity of the enzyme was also investigated and the system was applied as a modified carbon paste electrode to dopamine determination in pharmaceutical products. HRP adsorption onto PU-PEG nanoparticles followed the Langmuir isotherm model reaching an adsorption plateau at the concentration of 22.5μg HRPmg−1 nanoparticles. An estimation theoretical calculation was done regarding the number of proteins molecules immobilized per nanoparticle, based on the molecular weight of the protein and the nanoparticles size. It was found that about 4400 HRP molecules were adsorbed to one PU-PEG nanoparticle, which corresponds to about 45% of the surface area of the nanoparticle. Transmission electron microscopy (TEM) performed on immuno-gold labeled samples showed the HRP adsorbed onto the surface of PEGylated polyurethane nanoparticles. HRP-adsorbed PU-PEG nanoparticles dispersions retained 50% of the enzyme activity even after 50 days of storage. Square-wave voltammetry (SWV) experiments were carried out to investigate the performance of the modified carbon paste electrode containing HRP-adsorbed PU-PEG nanoparticles and the analytical curve was linear for dopamine concentrations from 1.7×10−5 up to 1.9×10−3molL−1 (r=0.9997), with a detection limit of 2.0×10−6molL−1. The recovery of dopamine from pharmaceutical samples ranged from 93% to 107% and the results obtained using the modified carbon paste electrode and those obtained by the official method are in agreement at the 95% confidence level.