Enzymatic Activity Of Glucose Oxidase Research Articles

Enzymatic Activity of Glucose Oxidase on Mesoporous TiO2:Mn Surfaces

Introduction: It is well known, that titanium dioxide (TiO2) nanoparticles can lead to the generation of reactive oxygen species (ROS) upon photoexcitation. Method: In this work, we investigated mesoporous surfaces based on TiO2 nanoparticles doped with 0.6-0.7% manganese (Mn), which showed reduced photoactivity and were based on the more stable rutile polymorph of titania. Result: In particular, we showed spectrophotometrically that the enzyme glucose oxidase (GOD) can be successfully adsorbed up to 80% while retaining its bioactivity in contact with the TiO2:Mn-based surface. Conclusion: We propose that this study could potentially give rise to biocompatible surfaces for biosensing applications.

Exploration of DNA Binding Proteins As a Versatile Tool for Fabrication of DNA-Protein Complexes and Its Application to Biosensing System

Introduction DNA-protein complexes composed of a DNA aptamer and an enzyme are useful to realize innovative biosensing systems; however, the stoichiometric and efficient fabrication method for DNA-protein complexes is yet to be developed. Chemical modification using amino acids on the surface of the protein is known as one irreversible conjugation way to fabricate DNA-protein complexes, but stoichiometric conjugation and retention of protein function to ensure high detection sensitivity are still a challenge. Therefore, in recent years, DNA binding proteins that covalently bind to specific DNA sequences have been utilized to fabricate DNA-protein complexes and applied as sensing elements. However, the binding efficiency to DNA of the DNA binding protein was low, which caused a decrease in detection sensitivity.Here, we introduce our challenge to utilize DNA binding proteins with high DNA binding ability as a versatile tool suitable for the fabrication of DNA-protein complexes. By fusing such DNA binding proteins to protein of interest, can be covalently linked to the fusion protein, leading to the preparation of stoichiometric DNA-protein complexes universally and conveniently (Fig. 1). We demonstrated the feasibility to construct a biosensing system of hemoglobin (Hb), composed of glucose oxidase (GOx) and anti-Hb aptamer as a representative example. Results & Discussion We explored the DNA binding proteins that can covalently bind to specific DNA sequences using the database to fabricate the stoichiometric and stable DNA-protein complex. Seventeen candidates were selected and classified into two families: HUH-endonuclease and uracil DNA glycosylase. We then selected three kinds of HUH-endonucleases: Rep domain of tomato yellow leaf curl virus (TYLCV), replication protein B (RepB), and conjugation transfer relaxase/helicase TraI (TraI). We expected them to have high recombinant production in the E. coli expression system because TYLCV and RepB have a relatively low molecular weight and TraI is derived from E. coli. We also selected UdgX because it is the only uracil DNA glycosylase known to form covalent bonds with DNA after removing uracil that aberrantly exists on DNA1). Thus, four DNA binding proteins with the ability to covalently bind to DNA were selected for further characterization.The binding ability of the four DNA binding proteins was compared. Each DNA binding protein was reacted with single stranded DNA (ssDNA) containing the corresponding recognition sequence and subjected to SDS-PAGE analysis. An ssDNA-dependent concentration band shift to higher molecular weight was observed for all DNA binding proteins upon covalent binding to each ssDNA. Based on the band quantification, we confirmed that UdgX had the highest binding efficiency and thus we used UdgX in following experiments.We attempted to detect Hb utilizing a biosensing element composed of GOx and anti-Hb aptamer fabricated using UdgX. We recently successfully integrated a protein coupling module, SpyCatcher/Tag system, into the universal fabrication method of sensing elements2-4). GOx and UdgX were also conjugated using SpyCatcher/SpyTag system, and then reacted with anti-Hb aptamer to fabricate aptamer-GOx complex. Hb has peroxidase activity and catalyzes chemiluminescence of luminol. Therefore, by using the aptamer-GOx complex, hydrogen peroxide produced by GOx is supplied to the Hb efficiently, and chemiluminescence of luminol is observed (Fig. 2).We confirmed homogeneous preparation of the aptamer-GOx complex by SDS-PAGE, and also the binding ability of aptamer and enzymatic activity of GOx. Using thus prepared DNA-protein complex, Hb was successfully detected with a high linearity in buffer and also in human serum without any washing procedures. The detection range was 4.0×10-3 to 32×10-3 g/L, this can meet the clinically required one (70 to 200 g/L) by diluting an actual sample. Because this system uses redox enzyme, it can be applied into the electrochemical measurement to achieve more rapid Hb detection. In this system, owing to integration of DNA binding protein and SpyCatcher/SpyTag system into sensing elements, a set of DNA aptamer and enzyme can be also easily exchanged, thus which has potential of various applications. References 1) P. B. Sang et al., Nucleic Acids Res., 43, 8452–8463 (2015).2) H. Kimura et al., Anal. Chem., 90, 14500-14506 (2018).3) H. Kimura et al., Biosens. Bioelectron., 175, 112885 (2021).4) D. Miura et al., Talanta, 234, 122638 (2021). Figure 1

Characterisation of physicochemical parameters and antibacterial properties of New Caledonian honeys.

Honey is an attractive natural product with various health benefits. A few honey-based commercial products have successfully been adopted in clinics to improve wound healing. However, screening of other potential sources of medical-grade honey, in particular, honeys from territories with high floral species diversity and high endemicity, is highly needed. The goal of this study was to characterise the physicochemical and antibacterial properties of New Caledonian honey samples (n = 33) and to elucidate the major mechanism of their antibacterial action. Inhibitory antibacterial activity of honeys against Staphylococcus aureus and Pseudomonas aeruginosa was determined with a minimum inhibitory concentration (MIC) assay. Enzymatic activity of glucose oxidase and the content of hydrogen peroxide (H2O2) in honey samples were analysed. Furthermore, total protein content of honeys together with their electrophoretic protein profiles were also determined in the study. The antibacterial efficacy of 24% of the tested honey samples was slightly superior to that of manuka honey with unique manuka factor 15+. The antibacterial activity of catalase-treated honey sample solutions was significantly reduced, suggesting that H2O2 is a key antibacterial compound of diluted honeys. However, the kinetic profiles of H2O2 production in most potent honeys at a MIC value of 6% was not uniform. Under the experimental conditions, we found that a H2O2 concentration of 150 μM in diluted honeys is a critical concentration for inhibiting the growth of S. aureus. In contrast, 150 μM H2O2 in artificial honey solution was not able to inhibit bacterial growth, suggesting a role of phytochemicals in the antibacterial activity of natural honey. In addition, the continuous generation of H2O2 in diluted honey demonstrated an ability to counteract additional bacteria in re-inoculation experiments. In conclusion, the tested New Caledonian honey samples showed strong antibacterial activity, primarily based on H2O2 action, and therefore represent a suitable source for medical-grade honey.

Enhancing Enzymatic Activity of Glucose Oxidase by Metal–Organic Framework Nanocarriers for Intensive Cascade Catalytic Antibacterial Therapy

Enhancing Enzymatic Activity of Glucose Oxidase by Metal–Organic Framework Nanocarriers for Intensive Cascade Catalytic Antibacterial Therapy

Bifunctional nanoprobe for dual-mode detection based on blue emissive iron and nitrogen co-doped carbon dots as a peroxidase-mimic platform

In this study, iron and nitrogen co-doped carbon dots (Fe/N@C-dots) were fabricated via one pot hydrothermal method. The as-synthesized Fe/N@C-dots exhibited unique properties such as high catalytic activity and fluorescence quantum yield (33.8%). The peroxidase-like activity of Fe/N@C-dots was used to detect H2O2 in the presence of phenol and 4-aminoantipyrine (4-AMAP) to yield a pink colored product (quinoneimine chromogen), which was measured at λmax = 505 nm (colorimetric method). Moreover, the pink colored product quenched the fluorescence of Fe/N@C-dots via inner-filter effect and static quenching (fluorometric method). The enzyme like activity of Fe/N@C-dots was cascaded with the enzymatic activity of glucose oxidase (GOx) to evaluate the practical application of Fe/N@C-dots to develop dual-channel sensor (fluorometric and colorimetric) for high selective and sensitive detection of glucose. It was found that the absorption intensities were increased linearly with glucose concentration in the range of 2.13–130 μM with LOD (S/N = 3) of 0.58 μM, while fluorescence intensities were decreased linearly after addition of glucose in the range of 1.33–140 μM with LOD (S/N = 3) of 0.36 μM. The Fe/N@C-dots/4-AMAP/phenol/GOx system along with the fluorometric method were applied efficiently to determine glucose in many samples including human serum, urine, and saliva with acceptable recoveries % in the range of 93.8–106.1% and RSD % not more than 3.7%, suggesting the reliability of the as-fabricated biosensor.

Chiral Control of Carbon Dots via Surface Modification for Tuning the Enzymatic Activity of Glucose Oxidase.

Chiral carbon dots (CDs) integrated the advantages of achiral CDs and the unique chiral property, which expand the prospect of the biological applications of CDs. However, the structure control and the origin of chirality for chiral CDs remain unclear. Herein, chiral CDs were obtained by thermal polymerization of chiral amino acids and citric acid, and their handedness of chirality could be controlled by adjusting the reaction temperature, which leads to different kinds of surface modifications. With aliphatic amino acids as a chiral source, all of the CDs that reacted at different temperatures (90-200 °C) have the same handedness of the chiral source. But with aromatic amino acids as a chiral source, CDs with maintained or inversed handedness compared with the chiral source could be obtained by adjusting the reaction temperature. Below a temperature of 120 °C, the chiral source was modified with CDs by esterification and transferred the handedness of chirality; at high temperatures (above 150 °C), which mainly connected by amidation accompanying with the formation of rigid structure generated by the π conjugation between the aromatic nucleus of chiral source and the carbon core of CDs, caused the inversing of the chiral signal. Further, we investigated the chiral effects of CDs on the glucose oxidase activity for a highly sensitive electrochemical biosensor.

Enzymatic activity of Glucose Oxidase from Aspergillus niger IPBCC.08.610 On Modified Carbon Paste Electrode as Glucose Biosensor

Glucose oxidase (GOx) has been developed as glucose sensor for measuring blood glucose level because of its specificity to glucose oxidation. This research aimed to determine kinetic parameters of GOx activity voltametrically and further test its potential as a glucose biosensor. GOx, in this research, was produced by local fungi Aspergillus niger IPBCC.08.610 which was isolated from local vine in Tarakan, East Borneo, Indonesia. GOx was immobilized with glutaraldehyde, which cross-linked onto modified carbon paste electrode (MCPE) nanofiber polyaniline. Intracellular GOx activity was higher than extracellular ones. Immobilized GOx used glutaraldehyde 2.5% and dripped on the surface of MCPE nanofiber polyaniline. MCPE have a high conductance in copper with the diameter of 3 mm. The concentration of glucose in the lowest concentration of 0.2 mM generated a current value of 0.413 mA while 2 mM of glucose induced a current of 3,869 mA value. Km and Imax of GOx in MCPE activities polyaniline nanofiber were 2.88 mM and 3.869 mA,respectively, with turnover (Kcat) of 13 s-1. Sensitivity was 1.09 mA/mM and response time to produce a maximum peak current was 25 seconds. Km value was then converted into units of mg/dL and obtained 56.4 mg/dL. GOximmo-IPB|MCPE electrode is potential to be able to detect blood glucose level in a normal condition and hypoglycemia conditions

Investigation of structural stability and enzymatic activity of glucose oxidase and its subunits

Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of β-d glucose utilizing molecular oxygen as an electron acceptor to produce d-glucono-1,5-lactoneand hydrogen peroxide, which has applications in food, biotechnology and medical industries. It was known that dimer form was considered to be active and monomer form has inactive conformation. However, there are no evidences at the molecular levels for Glucose oxidase (GOx) inactivation through dissociation. Here, using molecular dynamic simulation, it has been investigated for the first time that why dimer form of the enzyme is active. We have performed a series of molecular dynamics simulations at different forms of GOx (monomer and dimer with and without FAD cofactor). The analysis of tertiary structure showed that monomer is more unstable and has more deviation from the crystal structure. In contrast, dimer has a stable conformation during simulation. These results are in good agreement with experimental data about enzyme inactivation by dissociation. It was also found that when FAD is removed from monomer, it became more unstable in comparison with monomer containing cofactor. This shows essential role of FAD in both activity and stability of the enzyme. According to the MD simulation, enzyme inactivation is associated with changing in secondary structure at the interface. Interestingly, it was found that some secondary structures are destructed while some structures are formed in monomer upon dissociation. The analysis of active site structure during simulation revealed that both dissociation and release of the FAD influence on inactivation of GOx. This study provided novel insight to understand the mechanism of enzyme inactivation upon dissociation, which would be useful for rational enzyme design.

In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO43−-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H2S; the generated H2S instantly reacts with Cd2+ in the presence of Na3PO4 to give PO43−-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO43−-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h+, •OH, O2•− and a little H2O2, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO43−-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO43−-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

Evaluation of the Enzymatic Activity of Glucose Oxidase Immobilized on Multiwalled Carbon Nanotubes and on Controlled Pore Glass by Sequential Injection Analysis

ABSTRACTThe enzymatic activities of glucose oxidase immobilized on multiwalled carbon nanotubes and on controlled pore glass were evaluated. The evaluation was performed by optimizing a system of sequential injection analysis with amperometric detection using a 3-mm glassy carbon electrode as the working electrode. Glucose oxidase was immobilized on polyaniline-multiwalled carbon nanotubes using glutaraldehyde as a reagent. Transmission electronic microscopy showed the coupling of polyaniline on the multiwalled carbon nanotubes and the coupling of the enzyme on the polyaniline-multiwalled carbon nanotubes. The optimum values to determine the enzymatic activity of glucose oxidase on both supports by sequential injection analysis were 1 mmol L−1 pH 6 phosphate buffer, 25°C, glucose injection volume of 10 µL, reaction flow rate of 0.16 mL/min, an enzymatic reactor of 1 cm length x 0.2 cm i.d., 35 U of immobilized enzyme, and 0.8 V as the applied potential. The results showed a conditional Michaeles-Menten constant () of 5.38 (±0.23) mmol L−1 for the glucose oxidase-polyaniline-multiwalled carbon nanotube reactor and 1.30 (±0.06) mmol L−1 for the glucose oxidase-controlled pore glass reactor. The conditional maximum velocities () were 2.42 (±0.8) and 2.06 (±0.06) produced H2O2 min−1, respectively. The results suggest that the enzyme is more exposed to the substrate when it is immobilized on controlled pore glass.

Glucose oxidase-directed, instant synthesis of Mn-doped ZnS quantum dots in neutral media with retained enzymatic activity: mechanistic study and biosensing application.

Protein-directed synthesis of quantum dots (QDs) is a "greener" alternative to the current high-temperature and aqueous synthetic protocols, which provide water-soluble, biocompatible protein-functionalized QDs in one-pot. However, the protein activity in such synthetic schemes is a critical issue, since the synthetic conditions (for instance, high pH of the precursors, long time of synthesis, and disruption of disulfide bonds) are not suitable for retaining the activity (especially for enzymes). Herein, we present a facile and instant glucose oxidase (GOD)-directed strategy for the preparation of highly luminescent, phosphorescent Mn-doped ZnS (Mn-ZnS) QDs in one-step at room temperature and in neutral aqueous media. With such mild synthetic conditions, the enzymatic activity of GOD was totally retained. Furthermore, we also carried out GOD-directed synthesis of QDs with several other conditions that are reported in the literature. It turned out that the GOD enzymatic activity under these synthetic conditions was lower than that of the proposed protocol, indicating that mild synthetic conditions are the prerequisite for retaining the enzymatic activity. Importantly, the as-prepared GOD-mediated Mn-ZnS QDs exhibited high photostability, high salt tolerance and colloidal stability, and can be stored for months at 4 °C or 25 °C without changing their phosphorescent intensity and enzymatic activity. Via selective chemical modification, the exact functional groups (amino acid residues) of GOD in directing the synthesis of Mn-ZnS QDs were studied in detail. It turned out to be imidazole in histidine residues but not thiol in cysteine residues that directed the formation of Mn-ZnS QDs, and this was further confirmed with several other proteins for synthesis of Mn-ZnS QDs. The as-prepared GOD-capped Mn-ZnS QDs were employed as phosphorescent probes for background-free sensing of glucose in serum samples.

High-κ GdTixOy sensing membrane-based electrolyte–insulator–semiconductor with magnetic nanoparticles as enzyme carriers for protein contamination-free glucose biosensing

This paper reports an electrolyte–insulator–semiconductor (EIS) device featuring a novel high-κ GdTixOy sensing membrane for high-performance pH sensing and glucose biosensing. The effect of the annealing temperature (700, 800, or 900°C) on the sensing properties of the GdTixOy membranes was investigated. The GdTixOy EIS device annealed at 900°C exhibited the greatest pH sensing performance, including the highest sensitivity (62.12mV/pH), the smallest hysteresis voltage (5mV), and the lowest drift rate (0.4mV/h), presumably because of its well-crystallized GdTixOy structure. To overcome the problems typically encountered during the practical application of biosensors (e.g., protein adsorption; preservation of enzymatic activity), we employed Fe3O4-based magnetic nanoparticles (MNPs) as enzyme carriers. The adsorption of serum protein on the unmodified sensing membrane led to poor EIS-based pH sensing (r2=0.71); the performance was greatly improved, however, after attaching the MNPs to the sensing membrane, thereby blocking protein adsorption significantly (by 98%) and allowing excellent pH sensing (r2=0.99). Moreover, we prepared a hybrid configuration of the proposed GdTixOy membrane-EIS, with magnetically attached glucose oxidase-immobilized MNPs, for glucose biosensing. The use of MNPs as enzyme carriers effectively preserved the enzymatic activity of glucose oxidase, with 45.3% of the original enzymatic activity retained after 120h of storage at 4°C (compared with complete loss of the free enzyme's activity under the same storage conditions). In addition, the proposed biosensor exhibited superior detection sensitivity of 11.03mV/mM relative to that (8.17mV/mM) obtained using the conventional enzyme immobilization method. Finally, we established the accuracy of the proposed method for blood glucose measurement; gratifyingly, blood glucose detection was comparable with the high-sensitivity glucose quantification obtained using a commercial glucose assay kit.

Reusable sensor based on high magnetization carboxyl-modified graphene oxide with intrinsic hydrogen peroxide catalytic activity for hydrogen peroxide and glucose detection

We propose a new strategy to improve the enzyme stability, construction and sensitivity of a multifunctional sensor. An exfoliated graphene oxide sheet with carboxyl-long-chains (GO-CLC) was prepared in one step from primitive graphite via Friedel–Crafts acylation. Magnetic nanoparticles, glucose oxidase (GOD) and poly[aniline-co-N-(1-one-butyric acid) aniline] (SPAnH) were then incorporated to form an electrochemical film (SPAnH-HMGO-CLC-GOD) for the detection of hydrogen peroxide (H2O2) and glucose. The GO and Fe3O4 have intrinsic hydrogen peroxide catalytic activity and the activity will be enhanced by the combination of SPAnH coating and induces an amplification of electrochemical reduction current. This response can be used as a glucose sensor by tracing the released H2O2 after enzymatic reaction of bound GOD. Our sensor was linear within the range from 0.01mM to 1mM H2O2 and 0.1mM to 1.4mM glucose, with high sensitivities of 4340.6μAmM−1cm−2 and 1074.6μAmM−1cm−2, respectively. The relative standard deviations (RSD) were 5.4% for H2O2 detection and 5.8% for glucose detection. The true detecting range was 0.4–40mM for H2O2 and 4–56mM for glucose, which multiplied by 40-fold of dilution. This sensor based on the catalysis of organic SPAnH and the enzymatic activity of GOD can be used for both H2O2 and glucose sensing in potential clinical, environmental and industrial applications.

Quantifying Protein Adsorption and Function at Nanostructured Materials: Enzymatic Activity of Glucose Oxidase at GLAD Structured Electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

Kinetic Study of the Thermal Inactivation of Glucose Oxidase in the Presence of Denaturant and Stabilizer by Means of Bioelectrocatalysis Method

The thermal inactivation of glucose oxidase (GOD) in aqueous solution has been studied by the electrochemical method to follow the bioelectrocatalytic current due to the oxidation of glucose by GOD. Exponential time-dependent decrease in bioelectrocatalytic current, that is, the decrease in the enzymatic activity of GOD, was observed at given temperatures to determine the rate constant (k) of a simple inactivation process: GOD (active) → GOD (inactive). The ln[k] vs. T(-1) plots gave straight lines with all solution conditions tested, so that the resulting Arrhenius activation parameters including ΔH(‡) and ΔS(‡) can be compared with each other. In the 50 mmol/L phosphate buffer at 70°C, k was determined to be (6.6 ± 1.6)× 10(-4) s(-1), and ΔH(‡) and ΔS(‡) were calculated to be 202 ± 13 kJ mol(-1) and 282 ± 39 J K(-1) mol(-1), respectively. By addition of 3 mol/L guanidine hydrochloride, the k was increased to (4.7 ± 0.6)× 10(-3) s(-1), indicating that the denaturant accelerates the thermal inactivation. In this case, ΔH(‡) was significantly reduced. By addition of 1 g/L ε-poly-L-lysine, which may adsorb onto the GOD surface to reduce the local disorder, k was decreased to (1.8 ± 0.6)× 10(-4) s(-1). In this case, ΔS(‡) was reduced but ΔH(‡) was not decreased much. This can be used as an important indication for selection of the enzyme stabilizer in solution.

Light-Controlled Bioelectrochemical Sensor Based on CdSe/ZnS Quantum Dots

This study reports on the oxygen sensitivity of quantum dot electrodes modified with CdSe/ZnS nanocrystals. The photocurrent behavior is analyzed for dependence on pH and applied potential by potentiostatic and potentiodynamic measurements. On the basis of the influence of the oxygen content in solution on the photocurrent generation, the enzymatic activity of glucose oxidase is evaluated in solution. In order to construct a photobioelectrochemical sensor which can be read out by illuminating the respective electrode area, two different immobilization methods for the fixation of the biocatalyst have been investigated. Both covalent cross-linking and layer-by-layer deposition of GOD by means of the polyelectrolyte polyallylamine hydrochloride show that a sensor construction is possible. The sensing properties of this type of electrode are drastically influenced by the amount and density of the enzyme on top of the quantum dot layer, which can be advantageously adjusted by the layer-by-layer technique. By depositing four bilayers [GOD/PAH](4) on the CdSe/ZnS electrode, a fast-responding sensor for the concentration range of 0.1-5 mM glucose can be prepared. This study opens the door to multianalyte detection with a nonstructured sensing electrode, localized enzymes, and spatial read-out by light.

Effects of AC‐Electrolysis on the Enzymatic Activity of Glucose Oxidase

AbstractThe change in the activity of glucose oxidase subjected to an asymmetrical alternating current (AC) electric field is investigated via horseradish peroxidase (HRP)‐coupled bioassay. The effect of the amplitude, frequency and enzyme concentration have been shown to affect the enzyme activity towards glucose oxidation. The decrease in the enzyme activity is directly related to the change in pH and temperature of the GOx solution during AC electrolysis. The enzyme activity reduces with increasing amplitude, enzyme concentration and decreasing frequency. Results from UV‐vis, FT‐IR and UV CD spectroscopy showed that the AC treated GOx samples undergo structural modifications.

“Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H2O2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

Immobilization of enzymes at high load/activity by aqueous electrodeposition of enzyme-tethered chitosan for highly sensitive amperometric biosensing

Electrodeposition has been so widely used to immobilize biomacromolecules, and it is always an important topic to increase the load and activity of the immobilized biomacromolecules. We report here on a new, simple and rather universal method for the highly efficient immobilization of enzymes by aqueous electrodeposition of enzyme-tethered chitosan (CS) for sensitive amperometric biosensing. Glucose oxidase (GOx) is chosen here to examine the proposed protocol in detail. GOx was crosslinked to CS with low-concentration glutaraldehyde (GA, 0.080 wt%), and the electroreduction of added H 2O 2 increased the electrode-surface pH and triggered the electrodeposition of a GOx-GA-CS composite film. The GOx-GA-CS electrodeposition was monitored by an electrochemical quartz crystal microbalance and is theoretically discussed based on an electrogenerated base-to-acid titration model. The prepared first-generation enzyme electrode (CS-GA-GOx/Pt nano/Au) exhibits a current sensitivity as high as 102 μA mM −1 cm −2 at 0.70 V vs SCE, being 13 times that of the CS-GOx/Pt nano/Au prepared similarly but without the GOx-CS precrosslinking. UV–vis spectrophotometric determination of the GOx remains in the supernatant liquids after pH-induced CS precipitation suggested a high enzyme load in the GOx-GA-CS film, and amperometric measurements suggested a negligible decrease in the enzymatic activity of GOx after its reaction with the low-concentration GA. Also, the proposed protocol works well for the precrosslinking manner of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysulfosuccinimide activation, the water-electroreduction-triggered CS electrodeposition, the second-generation biosensing mode, a 5.0-μm-radius Pt ultramicroelectrode, and immobilization of alkaline phosphatase for phenyl phosphate biosensing. The proposed protocol of pretethering the target biomacromolecules to the electrodeposition precusor for immobilization of the biomacromolecule at high load/activity is recommended for wide applications.

Effect of refluxed silver nanoparticles on inhibition and enhancement of enzymatic activity of glucose oxidase

Glucose oxidase was immobilized on the surface of silver nanoparticles. The enzymatic activity of the glucose oxidase was determined by the refluxing time of silver nanoparticles. In a short refluxing time, the silver nanoparticles behaved to inhibit the enzymatic activity of the glucose oxidase. However, the inhibitory effect of silver nanoparticles reduced along with the refluxing time. And an enhancement effect appeared as the silver nanoparticles were refluxed in a certain period of time. When the refluxing time of silver nanoparticles was 3.0 h, the enzymatic activity of the immobilized glucose oxidase reached the maximum value, which was almost 4-fold as compared with that of the native glucose oxidase. Fluorescence and circular dichroism spectra indicated that the glucose oxidase on the surface of silver nanoparticles changed its conformation, and the conformation change depended on the refluxing time of silver nanoparticles, which was probably due to different interactions between glucose oxidase and silver nanoparticles.