Immobilization Of Horseradish Peroxidase Research Articles

Preparation of photo-crosslinkable cinnamate modified hyaluronic acid for immobilization of horseradish peroxidase

Abstract In this work, a photo-responsive hydrogel membrane based on cinnamate-modified hyaluronic acid (HA-CM) was developed and safely cross-linked under UV light curing. The obtained material was effectively utilized for immobilization of horseradish peroxidase (HRP) enzyme via encapsulation and entrapment strategy with efficiency above 95%. The prepared HA-CM biopolymer was investigated before the UV curing using instrumental and spectral techniques including Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). During the UV irradiation, the progress of the cross-linking reaction was monitored by the UV–vis light spectroscopy. In addition, when the photo-induced cross-linking had accomplished, the morphological appearance of the hydrogel membrane was recorded using a scanning electron microscope (SEM). The HRP immobilized in HA-CM membranes displayed remarkable stability against the environmental pH changes especially under alkaline media and shift the optimum pH to 8 compared to the free HRP, which exhibited the highest activity at pH 7. Also, the entrapped enzyme was able to preserve above 85% of its catalytic activity at higher temperature values where the free enzyme had deactivated by approximately 50%. Moreover, HA-CM-HRP maintained 87% of its activity after 10 sequential reuse cycles, which indicate the economic value of the employed immobilization strategy.

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.

Electrochemically reduced graphene oxide modified with electrodeposited thionine and horseradish peroxidase for hydrogen peroxide sensing and inhibitive measurement of chromium

Fabrication of hydrogen peroxide (H2O2) biosensor for determination of Cr(VI) based on the inhibition of horseradish peroxidase (HRP) activity is reported herein. The sensor platform was developed by using electrochemically reduced graphene oxide (ERGO) film modified GCE electrode. The thionine layer is deposited over ERGO layer by two step electrodeposition process. Immobilization of HRP over the thionine modified ERGO electrode has been done by mixing with bovine serum albumin (BSA) and using glutaraldehyde as a crosslinker. The cathodic responses of the immobilized HRP for H2O2 detection is monitored using cyclic voltammetry and linear sweep voltammetry method. Cr(VI) as a trace metal ions inhibitor to the enzyme activity was proportional to concentration in the range of 0.039–0.78 µM with the sensitivity of 4.41% inhibition µM−1 and the limit of detection is 0.02 µM (S/N = 3). The inhibition mechanism determined from Dixon and Cornish-Bowden plots was found to be uncompetitive. Good stability and reproducibility of the developed sensor could provide an excellent sensing platform for the determination of peroxide and heavy metals in environmental analyses and clinical testing.

Amidrazone modified acrylic fabric activated with cyanuric chloride: A novel and efficient support for horseradish peroxidase immobilization and phenol removal

In this study, hydrazine treated acrylic fabrics (polyacrylonitrile, PAN) activated with cyanuric chloride was developed as supporting material for horseradish peroxidase (HRP) immobilization. The immobilization of HRP onto the modified supporting material was achieved after being end-over-end incubated for 12 h. Field emission scanning electron microscopy and Fourier-transform infrared spectroscopy techniques were used to confirm the successful immobilization. Reusability experiment was performed to estimate the ability of the immobilized HRP to recover the reaction medium, in which it was observed to retain 78% of its original activity after 10 cycles. Relative to the soluble HRP, the optimum pH and temperature for the immobilized HRP were shifted to 7–7.5 and 50 °C, respectively. The kinetic parameters of guaiacol and H2O2 for the immobilized HRP were determined to be Km/Vmax = 57.61, 11.35 and Kcat/Km = 1.87, 1.86, respectively, while the values for the free form were Km/Vmax = 41.49, 6.23 and Kcat/Km = 1.87, 1.86, respectively. Compared to the soluble form, the immobilized HRP exhibited higher resistance toward metal ions and some organic solvents. For an application perspective. The immobilization of HRP using this procedure has the potential to be used for industrial application and wastewater treatment.

Immobilization of horseradish peroxidase on polyglycerol-functionalized magnetic Fe3O4/nanodiamond nanocomposites and its application in phenol biodegradation

In this study, Fe3O4/nanodiamond nanocomposites (MND) were synthesized by polyglycerol-mediated covalent bonding. The horseradish peroxidase (HRP) was successfully immobilized on PG layer of MND by interaction between functional groups of MND and HRP, where the HRP molecules became tridimensionally connected outside the MND. The physicochemical properties of MND were analyzed by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy measurements, vibrating sample magnetometer, Fourier transform infrared spectroscopy and zeta potential. The optimal conditions for the immobilization of HRP by MND are 7 mg/L, 35 °C and 3 h, respectively. Then, the effects of temperature, pH and storage time on the activity of immobilized enzyme and free enzyme were studied. The results showed that the relative activities of immobilized enzymes were higher than free enzymes at different temperatures, pH and storage time. In addition, the reusability experiments of immobilized enzymes showed that after six cycles, the immobilized HRP retained a relative activity of 75%. Applied to the removal of phenol, the effects of phenol concentration, H2O2/phenol molar concentration, immobilized HRP concentration and temperature on phenol removal were investigated. The results displayed that the removal efficiency of phenol reached a maximum when the phenol concentration was 75 mg/L, the ratio of H2O2 to phenol was 1, the immobilized HRP concentration was 0.25 mg/L and the temperature was 30 °C. The above results indicate that the immobilized HRP exhibits high removal efficiency and has great potential for removing phenol.

A Hydrogen Peroxide Biosensor from Horseradish peroxidase Immobilization onto Acrylic Microspheres

The sensitive and rapid detection of hydrogen peroxide is very important in the areas of clinical and environmental analyses. A sensitive and selective Horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) biosensor was developed based on acrylic microspheres. Hydrophobic poly(n-butyl acrylate-N-acryloxysuccinimide) [poly(nBA-NAS)] microspheres were synthesized using photopolymerization in an emulsion to form an enzyme immobilization matrix. The HRP enzyme was covalently immobilized onto the acrylic microspheres via the succinimide functionality. Field emission scanning electron microscope (FESEM) has been utilized to characterize the screen-printed carbon paste electrode (SPE) constructed from enzyme conjugated acrylic microspheres and gold nanoparticles (AuNPs) composite (HRP/nBA-NAS/AuNPs/SPE). Differential pulse voltammetry was used to assess the biosensor performance. The linear response range of the hydrogen peroxide biosensor obtained was from 1.0 × 10-2 to 1.0 × 10-10 M (R2 = 0.99) with the limit of detection (LOD) approximately at 1.0 × 10-10 M. This is an improvement over many hydrogen peroxide biosensors reported so far. Such improvement may be attributed to the large surface area provided by the acrylic microspheres as a matrix for immobilization of the HRP enzyme.

Competitive electrochemical immunosensor for maduramicin detection by multiple signal amplification strategy via hemin@Fe-MIL-88NH2/AuPt

Maduramicin (MD) is a type of monoglycoside polyether ionophore antibiotic that can effectively treat coccidiosis and facilitate animal growth. However, its extensive and excessive use brings potential risk to human health. Herein, an electrochemical immunosensor based on indirect competitive format was fabricated for analysis of MD residue in eggs by a multiple signal amplification system. Initially, Au nanoparticles were deposited onto glassy carbon electrode surface to load the coating antigen MD-BSA and to improve conductivity. Then the signal amplification platform was constructed by encapsulating hemin into Fe-MIL-88 NH2 metal-organic frameworks (hemin@MOFs), and then the obtained composites were decorated with AuPt nanoparticles. The synthesized hemin@MOFs/AuPt was not only used as a signal amplification mediator, but also utilized as a carrier for immobilization of horseradish peroxidase-conjugated affinipure goat anti-mouse antibody (Ab2-HRP) and horseradish peroxidase (HRP). The constructed hemin@MOFs/AuPt-Ab2-HRP bioconjugates could effectively amplify the current signal since hemin@MOFs, AuPt and HRP all exhibited high catalytic activity towards the hydrogen peroxide. Moreover, the established immunosensor showed high sensitivity and stability during the detection procedure. With the synergistic catalytic effect of hemin@MOFs, AuPt and HRP, a wide detection range of 0.1–50 ng mL−1 and a low detection limit of 0.045 ng mL−1 were achieved (S/N = 3), respectively. Ultimately, the developed method displayed excellent performance in practical applications, providing a promising probability to detect other veterinary drug residues to guarantee food safety.

An ultrasensitive electrochemical immunosensor for detection of sex hormone binding globulin

A novel electrochemical immunosensor was fabricated for detection of polycystic ovary syndrome (PCOS) by immobilizing horseradish peroxidase (HRP) labeled sex hormone binding globulin (SHBG) antibody (Ab) onto chitosan (CHIT), copper nanoparticles (CuNPs), iron nanoparticles (Fe3O4NPs) and graphene oxide nanoparticles (GrONPs) modified gold (Au) electrode. This HRP-SHBG Ab/CHIT/CuNPs/Fe3O4NPs/GrONPs modified Au electrode as working electrode, Ag/AgCl as standard electrode and Pt wire as auxiliary electrode were connected through potentiostat. The working electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectra (EIS) during its construction. The immunosensor possessed a dynamic linear range (0.01–250 nM) with a low limit of detection (LOD) of 0.01 nM. The evaluation study of immunosensor displayed a good analytical recovery of 98.03%, when SHBG antigen concentration was added in the serum sample. In addition, within and between batch coefficients of variation for working electrode were 0.115% and 0.111%, respectively. The immunosensor exhibited a correlation coefficient of (R2 = 0.99), which was underlying in agreement with standard method. The immunosensor lost 10% of its initial activity after its regular use for 180 days, while being stored at 4 °C.

Improving stability of biosensor based on covalent immobilization of horseradish peroxidase by γ-aminobutyric acid and application in detection of H2O2

A novel electrochemical biosensor for the determination of hydrogen peroxide (H2O2) has been fabricated through covalently immobilized horseradish peroxidase (HRP) on modified multi walled carbon nanotube (MWCNT) by γ-aminobutyric acid (GABA) on glassy carbon electrode. Using the electrochemical techniques, the electrochemical properties of the biosensor were specified. Cyclic voltammograms of the enzyme film at pH = 7.0 exhibited a pair of quasi-reversible redox peaks according to Fe(III/II) redox process of HRP. The biosensor exhibited good efficiency for finding H2O2 with a broad linear range from 2.0 × 10−7 M to 2.81 × 10−4 M and a detection limit of 0.13 μM. The surface coverage of active HRP, heterogeneous electron transfer rate constant and Michaelis-Menten constant of immobilized HRP were obtained 3.029 × 10−9 mol cm−2, 1.11 s−1, and 0.23 mM respectively. Similarly, the applicability of the suggested biosensor was examined by detecting H2O2 in human plasma samples and the average recovery was obtained as 100.50 ± 0.25. Moreover, the constructed biosensor presented a high stability, reproducibility and repeatability. Finally using docking and the molecular dynamics calculations, it was determined that GABA interacts with lysine residue and it causes HRP to be placed on the electrode with a specific orientation.

Enzyme Immobilization in MOF‐derived Porous NiO with Hierarchical Structure: An Efficient and Stable Enzymatic Reactor

AbstractMOF‐derived porous NiO with hierarchical structure (MHNiO) was prepared based on thermolysis of Ni metal organic framework (Ni‐MOF), which was used as carrier for the immobilization of horseradish peroxidase (HRP) and cytochrome C (Cyt c). The pore size of MHNiO was tuned as 11.8 nm to match the size of free enzyme so as to depress the aggregation of the enzymes in the pore. Meanwhile, the hierarchical structure allowed the substrates concentrated in the vicinity of the enzymes. The obtained enzymatic reactors exhibited better thermal stability, storage stability and reusability. For example, over 83 % of its original activity after 1 h incubation at 70 °C could be remained compared to 18.1 % or 38.4 % remained activity of free HRP and Cyt c, respectively; After 12 cycles of use, over 53 % of original catalytic activity of both the enzymatic reactor could be maintain. The enzymatic kinetic data (Km, Vmax, kcat) and thermodynamic data (Ka, ΔH, ΔS and ΔG) indicated that the improved catalytic performance was attributed to the affinity, selectivity and binding of enzyme to substrate. The two enzymatic reactors could be applied in the fast degradation of 2,4‐dichlorophenol and rifaximin in artificial wastewater, a complete degradation of 2 mg ⋅ mL−1 of 2,4‐dichlorophenol or 20 μg ⋅ mL−1 of rifaximin was achieved in only 20 min.

Enzyme immobilization in cage-like 3D-network PVA-H and GO modified PVA-H (GO@PVA-H) with stable conformation and high activity

In this work, polyvinyl alcohol hydrogel (PVA-H) and graphene oxide doped polyvinyl alcohol hydrogel (GO@PVA-H) with cage-like 3D network structure were synthesized by freeze/thaw recycling method and used for the immobilization of horse radish peroxidase (HRP). The variations of the network structures in PVA-H and GO@PVA-H were investigated under varied conditions. With the increasing PVA content (4–12%) and stirring time, both PVA-H and GO@PVA-H were tended to form more uniform pores and cage-like structure as well as smaller channel size. When GO was introduced, the stirring time was reduced to achieve appropriate mixing during the GO@PVA-H synthesis, compared to PVA-H with the same PVA content. HRP could be caged in PVA-H and GO@PVA-H and remained relatively high enzymatic activity. Experimental results showed that the structure and configuration of PVA-H and GO@PVA-H had significant effects on the enzyme accessibility and the conformational transformation of immobilized HRP. The structure analysis showed that the enzymatic conformation of HRP was changed by the immobilization under different conditions. The conformational transformation of immobilized HRP was restrained effectively in GO@PVA-H. After improving the structure of PVA-H and GO@PVA-H, the immobilized HRP acquired high dispersity, good accessibility and stable conformation, showed high-efficient activity similar as free HRP. Compared to PVA-H, not only the immobilization rate of HRP in GO@PVA-H but also the stability and reusability of HRP in the degradation of bisphenol A (BPA) was all improved. Therefore, PVA-H and GO@PVA-H were efficient in terms of storage and recycle of free enzymes in water treatment.

Immobilization of Horseradish Peroxidase on Fe3o4/au_go Nanoparticles to Remove 4-chlorophenols from Waste Water

Here we report the removal of 4-chlorophenols from waste water through the use of horseradish peroxidase (HRP) immobilized on Fe3O4/Au_GO nanoparticles. The large amount of surface functional groups on the support, which is coated with citric acid molecules, enables rapid enzyme immobilization through electrostatic interactions. HRP have been immobilized through physical adsorption on Fe3O4/Au_GO for 4-chlorophenols removal. The 4-chlorophenol degradation in presence of HRP immobilized on Fe3O4/Au_GO was 98 % after 180 min and at room temperature, with a reusability after three cycle of 95 %.

Towards a dual in-line electrochemical biosensor for the determination of glucose and hydrogen peroxide

Herein, we report the development of a sensitive and selective dual mode electrochemical platform for the detection of glucose and H2O2. The platform is based on tungsten and gold microwire electrodes decorated with gold nanoparticles (AuNPs) and colloidal platinum (colloidal-Pt), respectively. The nanostructured AuNPs electrode was used as a support matrix for the immobilization of horseradish peroxidase (HRP) and the colloidal-Pt served as the non-enzymatic glucose biosensor in the construction of a dual in-line electrochemical biosensor that provides a microenvironment for HRP and a pathway for analyte diffusion via the high surface area. The dual-mode approach allows for the simultaneous detection of glucose and H2O2. The glucose biosensor exhibited a dynamic linear range of 0.5 mM to 8 mM glucose. Linearity for H2O2 was up to 70 μM. Operational stability resulted in 75% of the initial biosensor response after 27 days. The dual in-line biosensor showed good sensitivity of 0.403 mA mM−1 cm−2 for glucose and 0.193 mA mM−1 cm−2 for H2O2 in addition to good anti-interference ability. Thus, the low-cost and simple dual in-line biosensor can be used for the real-time detection of glucose and H2O2 in the clinical, biological and environmental fields.

Ionic Liquid-Polypyrrole-Gold Composites as Enhanced Enzyme Immobilization Platforms for Hydrogen Peroxide Sensing.

In this work, three different aqueous solutions containing imidazole-based ILs with different alkyl chain lengths ([Cnmim]Br, n = 2, 6, 12) were adopted as the medium for the synthesis of ionic liquid-polypyrrole (IL-PPy) composites. Herein, the ILs undertook the roles of the pyrrole solvent, the media for emulsion polymerization of PPy and PPy dopants, respectively. The electrochemical performances of the three IL-PPy composites on a glassy carbon electrode (GCE) were investigated by electrochemical experiments, which indicated that [C12mim]Br-PPy (C12-PPy) composites displayed better electrochemical performance due to their larger surface area and firmer immobilization on the GCE. Further, C12-PPy/GCE were decorated with Au microparticles by electrodeposition that can not only increase the conductivity, but also immobilize sufficient biomolecules on the electrode. Then, the obtained C12-PPy-Au/GCE with outstanding electrochemical performance was employed as a horseradish peroxidase (HRP) immobilization platform to fabricate a novel C12-PPy-Au-HRP/GCE biosensor for H2O2 detection. The results showed that the prepared C12-PPy-Au-HRP/GCE biosensor exhibited high sensitivity, fast response, and a wide detection range as well as low detection limit towards H2O2. This work not only provides an outstanding biomolecule immobilization matrix for the fabrication of highly sensitive biosensors, but also advances the understanding of the roles of ILs in improving the electrochemical performance of biosensors.

Thiolated DAB Dendrimer-Gold Nanoparticles Self-Assembled Monolayer as Covalent Support for Direct Electrochemistry of HRP and Sensing Applications

A thiolated DAB dendrimer has been employed to bond gold nanoparticles of several sizes in order to obtain an electrocatalytic framework for the covalent immobilization and direct electrochemistry of horseradish peroxidase (HRP). This biosensor must represent the basis for developing a lot of oxidase-peroxidase bienzymatic biosensors and heavy metals biosensors based on the HRP inhibition. The kinetic study of the modified electrodes showed that the 5 nm and 16 nm gold nanoparticles are the most efficient to contact with the HRP active centre and the optimized biosensor allow to measure hydrogen peroxide at -0.3 V applied potential in linear ranges of 1-5000 and 1-140 or 140-5000 respectively with high sensitivities of 418.6 and 266.3 u A mM-1 cm-2 respectively too with low detection limits of 5 and 9 nM and fast response. The obtained apparent Michaelis-Menten constants, were 0.16 and 0.84 mM respectively. Both are significantly lower than the intrinsic K´M revealing the very high enzymatic efficiency of the developed devices.

Nanoporous Phyllosilicate Assemblies for Enzyme Immobilization.

Physical/chemical adsorption is well-known as a facile and effective method for enzyme immobilization, while ideal adsorbents with a high structural stability, high loading capacity, and low leaching ratio are still under exploration. In this study, nanoporous assemblies of two-dimensional (2D) copper phyllosilicate (L-CuSiO3) are prepared as an adsorbent to immobilize horseradish peroxidase (HRP) for phenol-containing wastewater treatment. Specifically, the robust chemical bonds of Si-O-Si and Si-O-Cu in L-CuSiO3 ensure its superior structural stability; the well-developed porous structure endows L-CuSiO3 assemblies with a high specific surface area of 611.7 cm3 g-1, which enables a fast and high enzyme loading of 140 mg g-1 within 4 h, and the well-distributed Cu(II) ions ensure the stable attachment of enzyme through Cu(II)-arginine (in HRP) coordination with a leaching ratio less than 10%. Meanwhile, the scaling assembly of L-CuSiO3 renders the resultant biocatalyst (HRP-loaded L-CuSiO3 assemblies) ease-of-recycling performance. Given the above features, the HRP-loaded L-CuSiO3 assemblies exhibit a better stability and 2-fold higher activity by contrast with HRP adsorbed on conventional mesoporous SiO2 and SiO2 nanoparticles, and it also acted as an efficient bioreactor in the application of catalytical removal of phenol pollutants from wastewater. Our L-CuSiO3 assemblies show great potential in immobilization of enzymes for industrial biocatalysis.

Photoelectrochemical biosensor for microRNA detection based on a MoS2/g-C3N4/black TiO2 heterojunction with Histostar@AuNPs for signal amplification

Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of microRNA-396a based on a MoS2/g-C3N4/black TiO2 heterojunction as the photoactive material and gold nanoparticles carrying Histostar antibodies (Histostar@AuNPs) for signal amplification. Briefly, MoS2/g-C3N4/black TiO2 was deposited on an indium tin oxide (ITO) electrode surface, after which gold nanoparticles (AuNPs) and probe DNA were assembled on the modified electrode. Hybridization with miRNA-396a resulted in a rigid DNA: RNA hybrid being formed, which was recognized by the S9.6 antibody. The captured antibody can further conjugate with the secondary IgG antibodies of Histostar@AuNPs, thereby leading to the immobilization of horse radish peroxidase (HRP). In the presence of HRP, the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 was accelerated, producing the insoluble product benzo-4-chlorohexadienone on the electrode surface and causing a significant decrease in the photocurrent. The developed biosensor could detect miRNA-396a at concentrations from 0.5 fM to 5000 fM, with a detection limit of 0.13 fM. Further, the proposed method can also be used to investigate the effect of heavy metal ions on the expression level of miRNAs. Results suggest that the biosensor developed herein offers a promising platform for the ultrasensitive detection of miRNA.

Immobilization of Horseradish Peroxidase on Multi-Armed Magnetic Graphene Oxide Composite: Improvement of Loading Amount and Catalytic Activity.

SUMMARYIn this study, a novel type of multi-armed polymer (poyltehylene glycol, PEG) magnetic graphene oxide (GO) composite (GO@Fe3O4@6arm-PEG-NH2) has been synthesized as a support for immobilization of horseradish peroxidase (HRP) for the first time. The loading amount of HRP was relatively high (186.34 mg/g) due to the surface of carrier material containing a large amount of amino groups from 6arm-PEG-NH2, but degradation rate of phenols was also much higher (95.4%), which is attributed to the synergistic effect between the free HRP (45.4%) and the support material of GO@Fe3O4@6arm-PEG-NH2 (13.6%). Compared with the free enzyme, thermal, storage and operational stability of the immobilized HRP improved. The immobilized HRP still retained over 68.1% activity after being reused 8 times. These results suggest that the multi-armed magnetic composite has good application prospect for enzyme immobilization.

Decolorization of a chromophore molecule with immobilized horseradish peroxidase

Decolorization of a chromophore molecule with immobilized horseradish peroxidase

An electrochemical enzymatic nanoreactor based on dendritic mesoporous silica nanoparticles for living cell H2O2 detection.

The selective and quantitative detection of cellular H2O2 is essential for understanding its roles in physiology and pathology. A new electrochemical H2O2 biosensor, fabricated by immobilizing horseradish peroxidase onto dendritic mesoporous silica nanoparticles (HRP/DMSNs), is employed for living cell H2O2 detection. Taking advantage of the large pore volume and highly accessible internal surface areas of DMSNs, HRP/DMSNs display higher enzymatic loading, better stability and bioactivity in comparison with HRP on nonporous silica nanoparticles (NSNs). Therefore, a HRP/DMSN modified GCE (HRP/DMSNs/GCE) shows attractive electrochemical performance for sensitive and selective detection of H2O2 in 0.1 M pH 7.0 PBS, with a low Kappm value of 11.48 μM and a low detection limit of 0.11 μM. In addition, HRP/DMSNs/GCE is successfully applied to detect H2O2 released from a PC12 cell triggered by ascorbic acid (AA). The detected H2O2 amount is close to the reported values. The developed biosensor has potential in the dynamic detection of the flux of H2O2 from living cells for further evaluation of oxidative stress in cells.