Modified Horseradish Peroxidase Research Articles

Magneto-Immunoassay for the Detection and Quantification of Human Growth Hormone.

Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L−1, R2 0.9831), low limit of detection (0.082 nmol L−1), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.

A nature-inspired colorimetric and fluorescent dual-modal biosensor for exosomes detection

As non-invasive biomarkers, exosomes are of great significance to diseases diagnosis. However, sensitive and accurate detection of exosomes still remains technical challenges. Herein, inspired by nature's “one-to-many” concept, we design a biosensor mimicking the cactus with numerous thorns to detect exosomes. The biosensor is composed of CD63 antibodies, resembling the roots of cactus, to capture exosomes, and the exosomes resemble the stems. Cholesterol-labeled DNA (DNA anchor) binding to streptavidin modified horseradish peroxidase (HRP) can insert into exosomes membrane, which seems the thorns. The readout signal is produced through HRP-catalyzed hydrogen peroxide (H2O2) mediated oxidation of 1,4-phenylenediamine (PPD) to form 2,5-diamino-NN′-bis-(p-aminophenyl)-1,4-benzoquinone di-imine (PPDox). The PPDox can quench fluorescence of fluorescein through inner filter effect (IFE), which provides fluorescent signal for exosomes detection. Based on this principle, the obtained exosomes solution is qualitatively and quantitatively analyzed by our biosensor, with the comparison to current standard methods by nanoparticle tracking analysis (NTA) and commercial enzyme-linked immunosorbent assay (ELISA) kit. The linear range is from 1.0 × 104 to 5.0 × 105 particles μL−1 with the limit of detection 3.40 × 103 particles μL−1 and 3.12 × 103 particles μL−1 for colorimetric and fluorescent assays, respectively. Meanwhile, our biosensor exhibits good selectivity, and can eliminate the interference from proteins. This dual-modal biosensor shows favorable performance towards analytical application in clinic samples, pushing one step further towards practical clinical use.

Aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) for electrochemical analysis of tumor biomarkers

Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3′-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5′-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can “sandwich” the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3′–OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

Ultrasensitive Electrochemical Aptasensor Based on Surface‐Initiated Enzymatic Polymerization

AbstractCoupled with the ability of fast and quantitative response, electrochemical aptasensors (EA) have great potential in the application of early diagnosis of cancer biomarker. In order to determine the rare biomarkers in the complicate clinical samples, scientists are making unremitting efforts towards improving the sensitivity and selectivity of EA. Herein, a "sandwich"‐structure electrochemical aptasensor (SEA) is developed for analysis of cancer biomarker carcino‐embryonic antigen (CEA). Two DNA aptamers are employed, one of which is thiolated at 3′‐terminal and immobilized onto the gold electrode as a capture probe, while the other one is served as signal probe. The two aptamers could "sandwich" the target and signal probe is then subjected to the terminal deoxynucleotidyl transferase (TdT)‐catalyzed incorporation of biotin labeled dNTPs into its 3′‐terminal. Thus the as‐generated long DNA oligo tails allow specific binding of numerous avidin modified horseradish peroxidase (Av‐HRP), resulting in enhanced peroxidase catalyzed electrochemical signals. This signal amplification strategy is termed as surface initiated enzymatic polymerization (SIEP). This SIEP amplified SEA has a detection limit of 10 pg·mL−1, indicating the outstanding amplification efficiency.

Nanoprobe-Initiated Enzymatic Polymerization for Highly Sensitive Electrochemical DNA Detection

Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated enzymatic polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3'-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5'-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a "sandwich" structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3'-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.

Measuring hydrogen peroxide due to water radiolysis using a modified horseradish peroxidase based biosensor as an alternative dosimetry method

H2O2 generated during water radiolysis was measured electrochemically as an alternative dosimetry method. A biosensor was fabricated by immobilising modified horseradish peroxidase (HRP) on a glassy carbon electrode (GCE) followed by evaluation of its analytical parameters. Anthraquinone 2-carboxylic acid was used to modify HRP. To assess sensor performance, phosphate buffer solutions were irradiated with 0.510Gy of gamma ray emitted from 60Co. The results showed that this sensor can detect low quantities of hydrogen peroxide in water radiolysis. Sensitivity, detection limit and linear range of the biosensor were 260nA/Gy, 0.392Gy and 0.5–5Gy, respectively. Long term stability studies showed that sensor responses were stable for at least a month. The cathodic peak current, as biosensor response, subsequently decreased to 20% of its initial value.

Biosensor For Detection Of Selective Anticancer Drug Gemcitabine Based On Polyaniline-gold Nanocomposite

Electrochemical biosensor is an effective tool for pharmaceutical analysis due to its simplicity, specificity, sensitivity, fast, cost- effective and repetitive measurements with miniaturized and portable devices. The paper illustrates the detail methodology for development of an amperometric biosensor based on polyaniline-gold nanocomposite film modified horseradish peroxidase for anticancer drug gemcitabine in bulk and in parenteral formulation. Scanning Electron Microscopy, Cyclic Voltammetry, Fourier Transform Infra Red Spectroscopy and Electrochemical Impedance Spectroscopic studies of the electrodes and after immobilizing of HRP shows the successful formation of a selectivity of the electrode. The proposed polyaniline-gold nano- composite based biosensor allow quantitation over the range 0.10 to 1.10 ngmL −1 with detection limit of 0.031 ngmL −1 , biosensor sensitivity of 2.934 µAng mL -1 has distinct advantages over other existing methods. Precision and accuracy were also checked and were within the limits. The procedure has been applied to the assay of the drug in dosage form with mean percentage recoveries of 99.00±0.08%. The suggested biosensor method can be successfully applied to the detection and determination of anticancer drug gemcitabine in different drug formulations. Copyright © 2015 VBRI press.

Preparation of graphene nanoplatelet–titanate nanotube composite and its advantages over the two single components as biosensor immobilization materials

A novel nanocomposite consisting of graphene nanoplatelets (GNPs) and titanate nanotubes (TNTs) have been synthesized successfully utilizing the hydrothermal method. The GNP–TNT composite was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and electrochemical impedance spectroscopy. The voltammetric characterization of GNP–TNT composite, pure GNPs and pure TNTs modified horseradish peroxidase (HRP) biosensors were conducted to select the most suitable electrode immobilization material for enzyme biosensors. The GNPs was firstly eliminated owing to its extremely high background charging current, distinct electrochemical interference from its surface functional groups and low signal to noise ratio. Next, the direct electron transfer of HRP on electrode and the catalytic current of HRP towards H2O2 was increased around 45% and 72% respectively on GNP–TNT composite modified electrodes compared with those on pure TNTs modified electrodes. GNP–TNT composite modified HRP biosensor also exhibited superiority over pure TNTs modified HRP biosensor in the analytical performance. The precision and stability study provided additional evidence for the feasibility of using GNP–TNT composite as electrode modification material.

Carbon felt-based bioelectrocatalytic flow-through detectors: Highly sensitive amperometric determination of H 2O 2 based on a direct electrochemistry of covalently modified horseradish peroxidase using cyanuric chloride as a linking agent

Horseradish peroxidase (HRP) was chemically modified using cyanuric chloride (CC) as a linking agent onto a carbon felt (CF), which is a microelectrode ensemble of micro carbon fiber (>7 μm, diameter) with a random three-dimensional structure. The resulting HRP-modified CF (HRP-ccCF) exhibited well-defined redox waves based on the HRP heme Fe III/Fe II redox couple at −0.23 V vs. Ag/AgCl (at pH 7.0), while the HRP-adsorbed CF (HRP-CF) showed no apparent redox couple in the same potential range, indicating that the chemical modification of HRP via CC facilitated the direct electron transfer (DET) between HRP and CF. The apparent heterogeneous electron transfer rate constant k s was estimated to be 35 s −1. Cyclic voltammetry and electrochemical impedance spectroscopy revealed that the interfacial properties (i.e., structure, morphology of enzyme-layer) of covalently modified HRP (HRP-ccCF) and physically adsorbed HRP (HRP-CF) are different, resulting in the difference in the electron transfer properties. The HRP-ccCF was successfully used as a working electrode unit in bioelectrocatalytic flow-through detector for highly sensitive amperometric determination of H 2O 2. Under the optimized conditions (i.e., applied potential, 0 V vs. Ag/AgCl; carrier flow rate, 3.25 ml/min; and carrier pH 7.0), the cathodic peak current of H 2O 2 linearly increased up to 3 μM (sensitivity, 1.94 μA/μM; the detection limit, 0.08 μM [S/N = 3]) with sample through-put of ca. 90 samples/h.

Dose rate determination of gamma rays emitted by Thallium-201 and technetium-99m using a modified horseradish peroxidase based biosensing system

In the presence of low energy gamma emitter radioisotopes of thallium-201 (201T1) or technetium-99m (99mTc), H2O2 was generated via radiolysis of water. The produced H2O2 was amperometrically determined using an anthraquinone 2-carboxylic acid modified horseradish peroxidase on glassy carbon electrode. In the presence of each radioisotope, the cathodic current produced due to the amperometric detection of H2O2, was designated as biosensor response. At the applied potential of -550 mV (vs. Ag/AgCl), the biosensor showed the sensitivities of 1.937 and 2.278 nA h ώGy-1 towards H2O2 produced by 201Tl and 99mTc respectively. Finally, the calibration curves for dose rate determination of 201Tl and 99mTc have been presented and the correlations between biosensor response to H2O2 and the gamma emitter dose rates for 201Tl and 99mTc are established.

Biosensing improvement of horseradish peroxidase towards hydrogen peroxide upon modifying the accessible lysines

A biosensor for detection of hydrogen peroxide was designed based on immobilization of anthraquinone 2-carboxylic acid modified horseradish peroxidase. Direct electrochemistry of the modified enzyme exhibited a quasi-reversible voltammogram with a formal potential of −459 mV. The surface charge transfer rate constant, k s, and the charge transfer coefficient, α c, for electron transfer between the glassy carbon electrode and the modified enzyme were estimated to be 3.165 s −1 and 0.455, respectively. The modified electrode exhibited a remarkable electrocatalytic activity towards hydrogen peroxide. Under the optimized experimental conditions, H 2O 2 could be detected in a linear concentration range from 2.65 × 10 −9 to 1.08 × 10 −7 M and a detection limit of 1.44 × 10 −9 M at S/N = 3. The response time was 6 s and when the electrode was stored at 4 °C, the biosensor was used repeatedly for 17 days with 10% deterioration in response.

Thermostability, solvent tolerance, catalytic activity and conformation of cofactor modified horseradish peroxidase

Artificial prosthetic groups, HeminD1 and HeminD2, were designed and synthesized, which contain one benzene ring and one carboxylic group or two carboxylic groups at the terminal of each propionate side chain of hemin, respectively. HeminD1 and HeminD2 were reconstituted with apo-HRP successfully to produce the two novel HRPs, rHRP1 and rHRP2, respectively. The thermal and solvent tolerances of native and reconstituted HRPs were compared. The cofactor modification increased the thermostability both in aqueous buffer and some organic solvents, and also enhanced the tolerance of some organic solvents. To determine the conformation stability, the unfolding of native and reconstituted HRPs by heat was investigated. T m was increased from 70.0 °C of nHRP to 75.4 °C of rHRP1 and 76.5 °C of rHRP2 after cofactor modification. Kinetic studies indicated that the cofactor modification increased the substrate affinity and catalytic efficiency both in aqueous buffer and some organic solvents. The catalytic efficiency for phenol oxidation was increased by ∼55% for rHRP1 in aqueous buffer, and it was also increased by ∼70% for rHRP1 in 10% ACN. Spectroscopic studies proved that the cofactor modification changed the microenvironment of both heme and tryptophan, increased α-helix content, and increased the tertiary structure around the aromatic residue in HRP. The improvements of catalytic properties are related to these changes of the conformation. The introduction of the hydrophobic domain as well as the retention of the moderate carboxylic group in active site is an efficient method to improve the thermodynamic and catalytic efficiency of HRP.

Increased thermal and organic solvent tolerance of modified horseradish peroxidase

Horseradish peroxidase (HRP) was modified by maleic anhydride and citraconic anhydride. The thermal and organic solvent tolerances of native and modified enzyme were compared. These chemical modifications of HRP increased their thermostability both in aqueous buffer and some organic solvents, and also enhanced their tolerances of some organic solvents. We have studied the unfolding of native and modified HRP by heat to determine the conformational stability. The temperature at the midpoint of thermal denaturation (T(m)) was increased upon modification. Both enthalpy change (DeltaH(m)) and entropy change (DeltaS(m)) for unfolding of modified enzyme at T(m) were decreased compared with native enzyme. Circular dichroism studies proved that these modifications changed the conformation of HRP. The improvements of stability are related to side chain reorientations of aromatics upon both modifications.

Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B(1g) distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B(2g)-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP.

Treatment of aqueous chlorophenol by phthalic anhydride-modified horseradish peroxidase

The reaction conditions of removal of chlorophenols from aqueous solution catalysed by phthalic anhydride (PA)-modified horseradish peroxidase were investigated. The optimal pH for chlorophenol removal decreased with increase in substituent number and it was not related to substituent position on aromatic ring. The optimum molar ratio of hydrogen peroxide to chlorophenol was 1.25. The effects of different substrates on phenolics removal were also investigated. The mixed phenolics were more easily removed. PA-modified horseradish peroxidase was more efficient in chlorophenol removal than native horseradish peroxidase either at low temperature or at high temperature. Aromatics with electron-withdrawing substituents (e.g. Cl) at the p-position favour removal over those with substituents at the o- or m-positions. The removal efficiency of 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol was almost equal and it was all lower than that of 4-chlorophenol. The kinetic constants for chlorophenol oxidation with native and PA-modified horseradish peroxidase were also determined. The kinetic data also proved the trend of removal of different chlorophenols with native or PA-modified horseradish peroxidase.

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies.

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO(1) and 36F(9) were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.

Structure of Poly(Ethylene Glycol)-Modified Horseradish Peroxidase in Organic Solvents: Infrared Amide I Spectral Changes upon Protein Dehydration Are Largely Caused by Protein Structural Changes and Not by Water Removal Per Se

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of α-helices and β-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160–169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.

Kinetic analysis of horseradish peroxidase “wiring” in redox polyelectrolyte–peroxidase multilayer assemblies

A hydrogen peroxide sensitive interface based on a self-deposited redox polyelectrolyte–peroxidase assembly was used as model system for investigation of the electron transfer between chemically modified horseradish peroxidase and the redox active centers of poly[(vinylpyridine)Os(bpy) 2Cl] redox polymer. The assembly was built up by sequential adsorption of the redox polymer and the peroxidase on gold electrodes modified with a self-assembled monolayer of 3-mercapto-1-propane sulfonic acid. Making use of a kinetic model, formulated for steady-state conditions, and of the rotating disc electrode technique it was possible to estimate: (i) the second order rate constant of reaction between the modified peroxidase and hydrogen peroxide ( 2.74×10 5 M −1 s −1 ) and (ii) the apparent turnover number of the oxidized enzyme regeneration by the osmium redox center ( 2.14 s −1 ), i.e., two parameters that reflect the efficiency of the electrical communication within the investigated interface.

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.

Dye bleaching and phenol precipitation by phthalic anhydride-modified horseradish peroxidase

The phenol precipitation and dye bleaching capabilities of phthalic anhydride–modified horseradish peroxidase C (PA–HRP) were compared with those of native HRP C and ethylene glycol- bis-(succinic acid N-hydroxysuccinimide ester)–modified HRP (EG–HRP) reported previously. Removal efficiency (percentage of phenol removed from solution under experimental conditions) was determined for native HRP and both modified forms. Removal efficiencies at 37 °C were very similar, with >95% removal in each case. Removal efficiencies were less at 70 °C overall (range 25–45%) but PA–and EG–HRP removed up to 50% more phenol than native HRP. The three HRP forms showed similar dye bleaching performance at 37 °C in the presence of H2O2 and accelerators (up to 86% colour removal). PA–and EG–HRP showed slightly greater bleaching abilities at 65 °C than native HRP for some of the dye/accelerator combinations tested. Modified HRPs performed better in 40% (v/v) mixtures of dioxane or dimethylformamide. © 2000 Society of Chemical Industry