Horseradish Peroxidase Solution Research Articles

Thread/paper- and paper-based microfluidic devices for glucose assays employing artificial neural networks.

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (μTPAD) and 3D microfluidic paper-based analytical device (μPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow-brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four-channel CMYK color data from 54 μTPAD and 160 μPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the μTPAD and μPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes.

Highly sensitive, stable and selective hydrogen peroxide amperometric biosensors based on peroxidases from different sources wired by Os-polymer: A comparative study

A comparison was made between two plant peroxidases, cationic horseradish peroxidase (HRP) and anionic tobacco peroxidase (TOP), combined with a highly cationic osmium polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2poly(N-vinylimidazole)10Cl]+2/+ ([Os(dmp)PVI]+/2+) to develop highly sensitive, stable and selective hydrogen peroxide biosensors. The two different plant peroxidases were individually immobilized onto graphite rod (G) electrodes by a three steps drop-casting procedure consisting of the subsequent deposition of an aqueous solution of ([Os(dmp)PVI]+/2+), followed by a solution of poly(ethyleneglycol) diglycidyl ether (PEGDGE), used as a cross linking agent and finally an aliquot of a solution of cationic HRP or anionic TOP to make HRP/PEGDGE/[Os(dmp)PVI]+/2+/G and TOP/PEGDGE/[Os(dmp)PVI]+/2+/G based electrodes, respectively. Electrochemical experiments were carried out to investigate the influence of the surface charge of the enzyme and the charge of the polymer on the efficiency of the electron transfer (ET) between the enzyme and the wiring redox polymer and the efficiency for electrocatalytic reduction of H2O2. In the case of HRP a decrease in the ET rate was observed due to the repulsion between this enzyme and the polymer, both positively charged, whereas with TOP there was an enhanced ET rate due to the attraction between the anionic enzyme and the cationic polymer. The effects of enzyme loading and pH were investigated. Both peroxidase modified electrodes exhibited a wide dynamic response range (1–500μM H2O2) and a low detection limit (0.3μM H2O2). The TOP based electrode showed a higher sensitivity (470nAμM−1cm−2) compared to that of the HRP based electrode (300nAμM−1cm−2) and an improved long-term stability (decrease in 17.3% upon 30days compared with 50% for HRP). Both enzyme electrodes showed a response time of 3s. The HRP based sensor was more sensitive to the presence of phenolic compounds acting as alternative electron donors, whereas the TOP based sensor was virtually interference free. Both HRP and TOP based electrodes were successfully tested in contact lens cleaning samples and real “spiked” samples from different sources such as tap water, milk and dairy products.

Synthesis and characterization of in situ gellable poly(glycerol sebacate)-co-poly(ethylene glycol) polymers

Hydrogels are widely used as implantable scaffolds and drug delivery carriers for biomedical applications. In particular, in situ cross-linkable hydrogels synthesized via enzyme-mediated reaction have received great attention in the field of injectable biomedical research as they have applications in minimally invasive procedures and have easily controllable physicochemical properties (e.g., gelation time, mechanical properties, etc.) under mild conditions. In this study, we synthesized poly(ethylene glycol) (PEG)-co-polymerized poly(glycerol sebacate) (PGS) polymers (PEG-co-PGS) capable of dissolving in aqueous environments and developed injectable hydrogel platforms via a horseradish peroxidase (HRP)-catalyzed cross-linking reaction. To induce in situ gelling, HRP-reactive phenol moieties (tyramine) were covalently conjugated to the PEG-co-PGS polymers, and hydrogel networks were formed in the presence of HRP and hydrogen peroxide (H2O2). The chemical structures of synthesized polymers were confirmed by 1H NMR spectroscopy, and the physicochemical properties of the hydrogels were assessed under varying concentrations of HRP and H2O2 solutions. The gelation time of PEG-co-PGS hydrogels ranged from 12 s to 237 s based on the HRP concentration (0.02-0.25 mg/mL), and the elastic modulus (16-41 Pa) depended on H2O2 concentration. In vitro cytocompatibility studies in human dermal fibroblasts revealed that PEG-co-PGS hydrogels were highly cytocompatible, with no negative effects on cell morphology and viability. In conclusion, our results suggest that PGS-based injectable hydrogels with multi-tunable properties and good cytocompatibility have tremendous potential as injectable scaffolds for tissue engineering applications.

Monitoring of microwave emission of HRP system during the enzyme functioning

Monitoring of microwave emission from aqueous solution of horseradish peroxidase (HRP) in the process of the enzyme functioning was carried out. For the monitoring, a system containing HRP, luminol and Н2О2 was employed. Microwave emission measurements were carried out in the 3.4-4.2GHz frequency range using the active and passive modes (active-mode and passive-mode measurements). In the active mode, excitation of the solution in the pulsed electromagnetic field was accomplished. In the passive mode, no excitation was induced. It appears that the passive-mode measurements taken in the course of the peroxidase reaction in the enzyme system have shown a 0.5°С increase of the microwave signal. Upon the active-mode measurements, taken in the same reaction conditions, the forced excitation of the solution has also led to the increase (by 2°С) of the level of the microwave signal – i.e. to its 4-fold enhancement compared to the signal obtained in passive-mode measurements.

Mixed thread/paper-based microfluidic chips as a platform for glucose assays.

A novel microfluidic thread/paper-based analytical device (μTPAD) to detect glucose through a colorimetric assay is described. The μTPAD was fabricated from nylon thread trifurcated into three channels terminating at analysis sites comprised of circular zones of chromatography paper, which have previously been spotted with glucose of different concentrations. A solution of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) is transported via capillary action to the analysis sites where a yellow-brown color is observed indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentrations. Both a flat platform constructed mainly of tape, and a cone platform constructed from tape and polyvinyl chloride, are described. Studies to quantitate glucose in artificial urine showed good correlation using the μTPAD.

Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on-column enzymatic assay.

A highly specific and sensitive method for glucose quantification in human serum samples based on on-column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter-microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2-[6-(4'-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H2 O2 ) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α-glucosidase activity and screening for its inhibitors.

Easily Fabricated Microfluidic Devices Using Permanent Marker Inks for Enzyme Assays.

In this communication, we describe microfluidic paper analytical devices (μPADs) easily fabricated from commercially available Sharpie ink permanent markers on chromatography paper to colorimetrically detect glucose using glucose oxidase (GOx). Here, solutions of horseradish peroxidase (HRP), GOx, and potassium iodide (KI)were directly spotted onto the center of the μPAD and flowed into samples of glucose that were separately spotted on the μPAD. Using an XY plotter (Roland DGA Corporation, Irvine, CA USA), several ink marks drawn in the paper act as the hydrophobic barriers, thereby, defining the hydrophilic fluid flow paths of the solutions. Two paper devices are described that act as independent assay zones. The glucose assay is based on the enzymatic oxidation of iodide to iodine whereby a color change from clear to brownish-yellow is associated with the presence of glucose. In these experiments, two designs are highlighted that consist of circular paper test regions fabricated for colorimetric and subsequent quantification detection of glucose. The use of permanent markers for paper patterning is inexpensive and rapid and does not require special laboratory equipment or technical skill.

A reusable electrochemical immunosensor fabricated using a temperature-responsive polymer for cancer biomarker proteins

In the present study, we describe a reusable electrochemical immunosensor for the repeated detection of cancer biomarkers using a single platform. The integration of a temperature-responsive polymer on the electrode surface enables easy manipulation of the biological sensing interface (i.e., addition of biotin, streptavidin, and antibody), thus allowing for temperature-induced regeneration and disruption of the interface architecture of the electrode surface. Using our immunosensor, we demonstrate sequential amperometric detection of three tumor markers: CA125, CEA, and PSA. Interestingly, greatly amplified signals are achieved by immersing the immunosensor in a solution of horseradish peroxidase (HRP) and antibody-labeled nanoparticles, resulting in a linear range of 0.0064 to 256U/mL, 1pg/mL to 100ng/mL, and 10pg/mL to 10ng/mL with a detection limit of 0.007U/mL, 0.7pg/mL, and 0.9pg/mL for CA125, CEA, and PSA, respectively. By alternating temperature, the immunosensor adsorbs and desorbs the biological elements without damage. Our proposed methodology can be expanded to measure other relevant biological species by repeated detection and thus has enormous potential for industrial and clinical applications.

Chemiluminescent Visualization for Evaluation of Gaseous Ethanol Distribution During ‘La France’ Pear Maturation

A 2-D visualization system for gaseous ethanol is constructed and tested in visualization of standard gaseous ethanol and maturation of fruit. This visualization system measures ethanol concentrations as intensities of chemiluminescence (CL) by luminol reaction induced by alcohol oxidase and a horseradish peroxidase-luminol-hydrogen peroxide system. Conversion of ethanol distribution and concentration to 2-D CL is conducted on an enzyme-immobilized mesh that contains alcohol oxidase, horseradish peroxidase and luminol solution. We visualize ethanol emission from a pear with maturation in which the concentration is typically at sub-parts per million (sub-ppm) level. Thus, a high-purity luminol sodium salt HG solution and an enhancer of fluorescence dye of eosin Y are optimized for the improvement of chemiluminescent intensity for visualization. The detection limit is improved sub-ppm using these solutions. Owing to high sensitivity, the emission of gaseous ethanol during La France pear (Pyruscommnis L.) maturation is successfully visualized and the intensity of each pixel is the reflection of ethanol concentrations. Therefore, this system is sufficiently useful for assessment of ethanol measurement for the maturation characteristic.

Sex Differences in the Number and Size of Motoneurons Innervating Rat Medial Gastrocnemius Muscle

The sex differences in the number and morphometric parameters of motoneurons in motor nuclei are poorly known. The aim of this study was to determine the differences in the number and size of alpha and gamma motoneurons of the medial gastrocnemius (MG) muscle in male and female Wistar rats. Retrogradely labelled cell bodies of motoneurons of 6 months old animals were studied following a bath of the proximal stump of the transected MG nerve in a horseradish peroxidase solution. The number and soma diameters of male and female MG motoneurons were determined from serial microscopic images of sections. The weight of the brain and spinal cord was on average 17% higher in males than in females. The mean number of motoneurons was 13% higher in males than in females and amounted to 94 and 83 motoneurons, respectively. In each case, the average soma diameters and cross-section areas of motoneurons in motor nucleus were distributed bimodally: motoneurons smaller than 27.5 μm in diameter were recognized as gamma and greater ones as alpha motoneurons. In males, the motor nucleus contained on the average 66 alpha motoneurons, whereas in females, 56 alpha motoneurons, that is the mean number of alpha motoneurons was 17% higher in males. Moreover, the soma diameters of gamma and alpha motoneurons were significantly bigger in males and the difference amounted 9 and 6%, respectively. It is concluded that the number as well as size of alpha and size of gamma motoneurons in the MG motor nucleus are greater in males.

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over-the-counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol-HRP-H2O2 system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol-HRP-H2O2 system. A putative enhancement mechanism for the luminol-H2O2-HRP-acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O-H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol-H2O2-HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%.

Horseradish peroxidase가 변성된 탄소복합 바이오센서 개발 및 전기화학적 H2O2분석

A sol-gel derived carbon composite electrodes (CCEs) were fabricated by mixing horseradish peroxidase (HRP), sol of tetraethoxysilane (TESO), and graphite powder. The HRP solution was added to the sol solution of TEOS, and then graphite powder was added to this mixture. The resulting carbon ceramic network effectively encapsulated HRP and shows a catalytic reduction starting at -0.2 V for H2O2. The optimum conditions for H2O2 determination have been characterized with respect to the enzyme loading ratio and pH. The linear range and detection limit of H2O2 detection were from 0.2 mM to 2.2 mM and 0.035 mM, respectively. The common electroactive interferences such as ascorbic acid, ace- taminophene, and uric acid were not affected upon the response to H2O2 at the HRP biosensor due to low detection potential.

Facile synthesis of new periodic mesoporous organosilica and its performance of immobilizing horseradish peroxidase

A new class of periodic mesoporous organosilicas (PMOs), which contains various hydrophilic groups of –OH, –O– and –S–, is successfully synthesized with novel bridged organosilica precursor under the acid-catalyzed and inorganic-salt-assisted conditions. These hybrid materials possess the ordered mesostructure with uniform pore size distributions, thick framework walls, thermal stability and specific functional groups; and they are carefully characterized by XRD, N2 adsorption, TGA, FT-IR, TEM and NMR spectroscopies. Moreover, horseradish peroxidase (HRP) is immobilized on these new PMO composites for the first time, and their enzyme activity in the oxidation of 1,3,5-trimethylbenzene (TMB) is elevated in the range of 25–59% compared with that of free HRP in solution. Again, the HRP immobilized samples can be reused for many cycles, resulting from the interfacial activation of the enzyme when it is attached to the surface of hybrid hosts where is filled with organic groups and hydrophobic parts.

In situ gellable sugar beet pectin via enzyme-catalyzed coupling reaction of feruloyl groups for biomedical applications

In situ gellable hydrogels are more attractive in many biomedical and biopharmaceutical applications than pre-formed hydrogels because they can be implanted simply by injection and allow homogeneous incorporation of bioactive materials. In this study, the potential suitability of in situ gellable sugar beet pectin (SBP) for biomedical and biopharmaceutical applications was investigated. SBP aqueous solution gelled within 1 min after addition of appropriate amounts of horseradish peroxidase (HRP) and H₂O₂ via HRP-catalyzed oxidative coupling reaction of feruloyl groups on SBP molecules. The resultant gels gradually degraded under simulated physiological condition. L929 fibroblast cells encapsulated in the gels were scarcely damaged during the gelation process. A subcutaneously injected mixture of SBP, HRP and H₂O₂ solutions successfully gelled, and the gel did not induce necrosis in the surrounding tissue 1 week after implantation. These results demonstrate that the in situ gellable SBP gels are useful for biomedical and biopharmaceutical applications.

Carbon-felt-based Bioelectrocatalytic Flow-detectors: Optimization of the Adsorption Conditions of Horseradish Peroxidase and Thionine onto Carbon-felt for Highly Sensitive Amperometric Determination of H2O2

Horseradish peroxidase (HRP) and thionine (Th) were co-adsorbed onto a porous carbon-felt (CF) from a mixed solution of HRP and Th to fabricate a CF-based bioelectrocatalytic flow-detector for H(2)O(2). The resulting HRP and Th co-adsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a highly sensitive amperometric flow-H(2)O(2)-biosensor. Differing from the ordinary mediator-based HRP biosensors, which detect mediated currents (i.e., the reduction current of the oxidized form of the mediators), the present HRP/Th-CF-based H(2)O(2)-biosensor directly detects the reduction current of the oxidative HRP-intermediates (i.e., compound I and II) based on a direct electron transfer (DET) between a HRP-redox active heme center and the CF electrode, because co-adsorbed Th together with HRP facilitate the DET. Various adsorption conditions (i.e., HRP concentration, Th concentration, ionic strength, pH, adsorption time) were optimized to obtain excellent sensor performances. When an air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at 3.25 ml/min and the applied potential of - 0.05 V vs. Ag/AgCl, the HRP/Th-CF-based flow-H(2)O(2)-biosensor exhibited excellent analytical performances (i.e., sensitivity, 3.55 µA/µM; linear range, 0.1 to 10 µM; lower detection limit, 0.03 µM; K(m)(app), 36.2 µM; I(max), 158 µM; sample throughput, ca. 60 samples/h).

Elevating enzyme activity through the immobilization of horseradish peroxidase onto periodic mesoporous organosilicas

Horseradish peroxidase (HRP) is immobilized on periodic mesoporous organosilicas (PMO) for the first time, and the enzyme activity improved, especially on MI100, prepared with 100% organic silica precursor, the enzyme activity is enhanced 110% compared with free HRP in solution. Besides, the stability of immobilized HRP to lower pH values or the denaturing agent urea is enhanced, resulting from interfacial activation of the enzyme when it is attached to a surface filled with organic groups and hydrophobic parts. The thermal stability of immobilized HRP on PMO is also increased and, moreover, the HRP immobilized samples can be reusable for many cycles.

Carbon-felt-based Bioelectrocatalytic Flow-detectors: Role of Ultrasound Irradiation during the Adsorption of Horseradish Peroxidase and Thionine for a Highly Sensitive Amperometric Determination of H<sub>2</sub>O<sub>2</sub>

This study reports on the first example, to our knowledge, of the usefulness of an ultrasound (US)-irradiation during an enzyme adsorption step, for enhancing the performance of a redox-enzyme-based amperometric biosensor. Horseradish peroxidase (HRP) and thionine (Th) were coadsorbed from a mixed aqueous solution of HRP and Th onto a carbon-felt (CF) under US-irradiation for 5 min with an ultransonic bath operating at 40 kHz frequency and 55 W of electric power output. The resulting HRP and Th-coadsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a bioelectrocatalytic flow-detector for hydrogen peroxide (H(2)O(2)), which detects the cathodic peak currents based on the direct (unmediated) reduction of oxidized HRP intermediates at 0 V vs. Ag/AgCl. Compared with ordinary adsorption without US-irradiation, US-irradiation during the HRP adsorption step was effective to obtain highly sensitive peak current responses to H(2)O(2). The measurements of electrochemical impedance spectroscopy and cyclic voltammetry suggested that the adsorption of HRP and Th under the US-irradiation provides a suitable interfacial microenvironment for a favorable orientation and conformation of an enzyme with active site available for both substrates and the electrode, which results in larger bioelectrocatalytic activity. The peak currents for H(2)O(2) increased up to 3 × 10(-6) M (sensitivity, 4.72 µA/µM) with a lower detection limit of 2 × 10(-8) M (S/N = 3; current noise level, 0.03 µA).

Chemiluminescent Detection of Horseradish Peroxidase Using an Integrated Amorphous Silicon Thin-Film Photosensor

A hydrogenated amorphous silicon p-i-n photodiode fabricated on a glass substrate is used for the integrated, real-time detection of biochemiluminescent reactions. The light emission at 425 nm resulting from the oxidation of luminol catalyzed by the enzyme horseradish peroxidase (HRP) is detected. This enzyme is commonly used as a label attached to biomolecules such as proteins and nucleic acids. In a previous publication (Pimentel, et al., 2007), HRP was detected in solution with a sensitivity of 2 nM. In this work, HRP in solution was detected with sensitivity of 10 pM. In addition, HRP adsorbed in the surface was detected with sensitivity in the range of 1 fmol·cm-2. Antibody-antigen recognition was successfully detected using antibodies labeled with HRP.

Parasympathetic innervation of the initial segments of the large intestine in cats

The locations and morphometric characteristics of efferent parasympathetic neurons in the dorsal motor nucleus of the vagus nerve and the cruciform parasympathetic nucleus of the spinal cord, innervating the area of the ileocecal sphincter and the ascending and transverse segments of the colon, were studied. Horseradish peroxidase solution was injected beneath the serous membranes of these parts of the intestine in urethane-anesthetized cats. After 48 h, animals were subjected to transcardiac perfusion with a fixative mixture and sections of the medulla oblongata and spinal cord were prepared and processed by the Mesulam method. The results showed that all these parts of the large intestine received parasympathetic innervation from neurons in the ventrolateral part of the dorsal motor nucleus, which were uniform in terms of their morphometric characteristics. The number of neurons of this group sending axons to the ileocecal area was greater than the number of neurons innervating the ascending colon. A second group of neurons, which were smaller cells, was located in the same part of the nucleus and innervated the transverse colon. The transverse colon also received innervation from neurons in the cruciform parasympathetic nucleus of the spinal cord.

Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.