Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO₂ nanoparticles (nano-TiO₂) on a gold electrode. The nano-TiO₂ can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet–visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H₂O₂ was from 2.3 × 10⁻⁶ to 2.4 × 10⁻³ M with a detection limit of 7.0 × 10⁻⁷ M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.     

Glucose biosensor based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite

The direct immobilization of glucose oxidase (GOD) on TiO₂/SiO₂ nanocomposite and its application as glucose biosensor were investigated. The room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite can be quenched by hydrogen peroxide (H₂O₂). The detection of glucose may be accomplished by monitoring the formation of hydrogen peroxide which generated in the oxidation process of glucose with the catalysis of GOD. To our surprise, by using a 96-hole polyporous plate accessory of fluorescence spectrophotometer, the biosensor exhibits excellent linear response to glucose concentrations ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻² M with a detection limit of 1.2 × 10⁻¹⁰ M. The TiO₂/SiO₂ nanocomposite can be used as both supporting material and signal transducer. The phosphorescence intensity and color of the biosensor change obviously and even could be observed with naked eyes by continuous addition of glucose. Based on the room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite, a new method of solid substrate-room-temperature phosphorimetry (SS-RTP) for glucose determination is proposed. A glucose biosensor was fabricated with wide determination concentration range, low detection limit, high sensitivity, and fast response time. And the biosensor has been successfully applied to the determination of glucose in human blood serum. The coacervation of GOD enzyme and its interaction with TiO₂/SiO₂ nanocomposite enlarge the surface area and enhance the chemical stability of GOD. The nice biocompatibility, large surface area, good chemical stability and nontoxicity of the TiO₂/SiO₂ nanocomposite have made this material suitable for functioning as biosensor.     

Glucose biosensor based on titanium dioxide-multiwall carbon nanotubes-chitosan composite and functionalized gold nanoparticles

In this paper, a new glucose biosensor was prepared. At first, Prussian blue (PB) was electrodeposited on a glassy carbon electrode (GCE) modified by titanium dioxide-multiwall carbon nanotubes-chitosan (TiO₂-MWNTs-CS) composite, and then gold nanoparticles functionalized by poly(diallyldimethylammonium chloride) (PDDA-Au) were adsorbed on the PB film. Finally, the negatively charged glucose oxidase (GOD) was self-assembled on to the positively charged PDDA-Au. The electrochemical performances of the modified electrodes had been studied by cyclic voltammetry (CV) and amperometric methods, respectively. In addition, the stepwise fabrication process of the as-prepared biosensor was characterized by scanning electron microscopy. PDDA-Au nanoparticles were characterized by ultraviolet–vis absorption spectroscopy and transmission electron microscopy. Under the optimal conditions, the as-prepared biosensor exhibited a good response performance to glucose with a linear range from 6 μM to 1.2 mM with a detection limit of 0.1 μM glucose (S/N = 3). In addition, this work indicated that TiO₂-MWNTs-CS composite and PDDA-Au nanoparticles held great potential for constructing biosensors.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Molecular docking,a tool to determine interaction of CuO and TiO2 nanoparticles with human serum albumin

BackgroundWe study the human serum albumin (HSA) protein-CuO nanoparticle interaction to identify the specific binding site of protein with CuO nanoparticles by molecular docking and compared it with HSA-TiO₂ nanoparticle interaction.MethodsThe protein structural data that was obtained using Autodock 4.2.ResultsIn case of CuO np-HSA interaction, the distances from the centre of Subdomain IIIA to Arg-472 is 2.113 Å and Lys 475, Glu 492, Ala 490, Cys 487, Ala 490 are the bound neighbouring residues with Lys 475, Glu 492 at aliphatic region. The binding energy generated was ?1.64 kcal mol^?1. However, for TiO₂ nanoparticle, the binding region is surrounded by Arg 257, Ala 258, Ser 287, His 288, Leu 283, Ala 254, Tyr 150 (subdomain II A) as neighbouring residue. Moreover, Glu 285, Lys 286 forms aliphatic grove for TiO₂-HSA, Ser-287 at the centre region form hydrogen bond with nanoparticle and Leu 283, Leu 284 forming hydrophopobic grove for TiO₂ nanoparticle-HSA interaction. The binding energy generated was ?2.47 kcal mol^?1.ConclusionsAnalysis suggests that CuO bind to suldow site II i.e subdomain III A of HSA protein where as TiO₂ nanoparticle bind to suldow site I i.e subdomain IIA of HSA protein.General significanceThe structural information that derives from this study for CuO and TiO₂ nanoparticles may be useful in terms of both high and low-affinity binding sites when designing these nanoparticles based drugs delivery system.     

Electrochemiluminescent pH sensor measured by the emission potential of TiO2 nanocrystals and its biosensing application

This work reports for the first time a potential‐based nano‐electrochemiluminescent (ECL) pH sensor, using anatase TiO₂ nanocrystals (NCs) as the ECL probe. The first ECL peak potential of the TiO₂ NCs shifted negatively with increasing pH, showing a linear range from −0.47 V (vs Ag/AgCl) at pH 3 to −1.06 V at pH 10. This phenomenon was attributed to the absorption of ‘potential‐determining ions’ of OH⁻ on the surface of TiO₂ NCs, leading to larger impedance of the electron injection. Other common ‘potential‐determining ions’, such as phosphate, induced a slight potential shift of 0.03 V at a concentration of 0.1 M. Using urease as an enzyme model, a urea biosensor was developed by the simultaneous modification of urease and TiO₂ NCs on indium–tin oxide (ITO) electrodes. The biosensor, measured on the basis of the pH increase caused by the enzyme catalysis reaction, had a linear range of 0.01–2.0 mM, with a potential shift of 0.175 V. The as‐prepared pH sensor, which has simple construction procedures and acceptable sensitivity and selectivity, may provide new avenues for the construction of ECL bioanalytical methodologies. Copyright © 2014 John Wiley & Sons, Ltd.     

Activation of nylon net and its application to a biosensor for determination of glucose in human serum

A glucose biosensor using a glucose oxidase (GO_x)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O₂) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O₂ in oxidation of glucose by the membrane bound GO_x. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.     

Direct electrochemistry of hemoglobin based on chitosan–ionic liquid–ferrocene/graphene composite film

This paper described an ingenious approach for the fabrication of a promising biosensor, hemoglobin (Hb)/chitosan (Chit)–ionic liquid (IL)–ferrocene (Fc)/graphene (Gr)/glassy carbon electrode (GCE), that exploited the synergistic beneficial characteristics of Fc, Gr and IL for Hb. The proposed biosensor showed a strong electrocatalytic activity toward the reduction of H₂O₂, which could be attributed to the favored orientation of Hb in the well-confined surface as well as the high electrical conductivity of the resulting Chit–IL–Fc/Gr inorganic hybrid composite. The developed biosensor exhibited a fast amperometric response (2 s), a good linear response toward H₂O₂ over a wide range of concentration from 50 μM to 1200 μM, and a low detection limit of 3.8 μM. The apparent Michaelis–Menten constant (K_m) of Hb on the composite medium was 0.16 mM, showing high bioelectrocatalytic activity of immobilized protein toward H₂O₂ reduction. High sensitivity and stability, technically simple and possibility of preparation at short period of time are of great advantages of the developed biosensors.     

Titanium Dioxide (TiO2) Nanoparticles Preferentially Induce Cell Death in Transformed Cells in a Bak/Bax-Independent Fashion

While the cytotoxic effects of titanium dioxide (TiO₂) nanoparticles have been under intense investigation, the molecular mechanisms of this cytotoxicity remain unknown. Here we investigated the influence of oncogenic transformation and a major apoptotic signaling pathway on cellular responses to TiO₂ nanoparticles. Isogenic wild-type (WT) and apoptosis-resistant (Bak^−/−Bax^−/−) cell lines with and without tumorigenic transformation were examined. TiO₂ nanoparticles preferentially reduced viability of tumorigenic cells in a dose-dependent fashion compared with their untransformed counterparts. Importantly, the elevated cytotoxicity of TiO₂ nanoparticles was independent of a major Bak/Bax-dependent apoptosis pathway. Because transformation does not affect cellular fluid-phase endocytosis or nanoparticle uptake, it is likely that the increased cytotoxicity in tumor cells is due to the interaction between TiO₂ nanoparticles and the lysosomal compartment. Overall, our data indicate that TiO₂ nanoparticles induce cytotoxicity preferentially in transformed cells independent of a major apoptotic signaling pathway.     

Quantification of titanium from TiO2 particles in biological tissue

This study presents the development of a strategy for the quantification of titanium from titanium dioxide polydisperse particles (TiO₂) in dry biological tissue. Calf liver was chosen as laboratory testing material. The challenge was to (i) obtain a complete mineralization of the solid material (biological tissue and TiO₂) and (ii) ensure the accuracy of the determined concentrations with a sufficient sensitivity. Mineralization was performed using a mixture of concentrated nitric and hydrofluoric acids. Atomic mass spectrometry associated with light-scattering technique was used to control the physical state (dissolved and particle forms) of titanium and reliably estimate the total titanium concentration in calf liver. The monitoring of ⁴⁶Ti and ⁴⁹Ti, operating in helium collision/reaction cell mode, and using external calibration with internal standard addition, allowed the quantification of Ti while removing isobaric interferences. The limit of detection and quantification were 0.7 and 2.3 μg (Ti) g⁻¹ (tissue) respectively. The mean analytical recovery over the whole procedure was (103 ± 6)% in a range of concentrations from LOD to 200 μg(Ti) g⁻¹ (tissue).     

Electrogenerated chemiluminescence ethanol biosensor based on alcohol dehydrogenase functionalized Ru(bpy)3 doped silica nanoparticles

An ethanol biosensor, based on the electrogenerated chemiluminescence of Ru(bpy)₃²⁺-doped silica nanoparticles (RuSiNPs), was investigated in this study. The biosensor was a modified glassy carbon electrode, where alcohol dehydrogenase was crosslinked to RuSiNPs, and then immobilized on the electrode surface using chitosan. The results indicated that the biosensor exhibited excellent performance during ethanol determination with a wide linear range (10⁻⁷ to 10⁻² M), low detection limit (5.0 × 10⁻⁸ M) and good stability.     

[Cu(phen)2] acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)₂]²⁺, where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)₂]²⁺ acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)₂]²⁺ were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10⁻¹² to 1 × 10⁻⁶ M with a detection limit of 1.99 × 10⁻¹³ M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.     

A Simple and Sensitive SPRS Method for Trace H2O2 Based on the TiO2+ Complex and Gold Nanoparticles Aggregation Reactions

In the medium of H₂SO₄ and in the presence of TiO²⁺, gold nanoparticles in size of 10 nm exhibited a weak surface plasmon resonance scattering (SPRS) peak at 775 nm. Upon addition of trace H₂O₂, the yellow complex [TiO(H₂O₂)]²⁺ formed that cause the gold nanoparticles aggregations to form bigger gold nanoparticle clusters in size of about 900 nm, and the SPRS intensity at 775 nm (I) enhanced greatly. The enhanced intensity ΔI was linear to the H₂O₂ concentration in the range of 0.025–48.7 μg/mL, with a detection limit of 0.014 μg/mL H₂O₂. This SPRS method was applied to determining H₂O₂ in water samples with satisfactory results.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Construction of protein-modified TiO2 nanoparticles for use with ultrasound irradiation in a novel cell injuring method

Recently, our group discovered an alternative titanium dioxide (TiO₂) activation method that uses ultrasound irradiation (US/TiO₂) instead of ultraviolet irradiation. The pre-S1/S2 protein from hepatitis B virus, which recognizes liver cells, was immobilized to the surface of TiO₂ nanoparticles using an amino-coupling method. The ability of the protein-modified TiO₂ nanoparticles to recognize liver cells was confirmed by surface plasmon resonance analysis and immuno-staining analyses. After uptake of TiO₂ nanoparticles by HepG2 cancer cells, the cells were injured using this US/TiO₂ method; significant cell injury was observed at an ultrasound irradiation intensity of 0.4 W/cm². Together with these results, this strategy could be applied to new cell injuring systems that use ultrasound irradiation in place of photodynamic therapy in the near future.     

The effect of UV radiation in the presence of TiO2-NPs on Leishmania major promastigotes

Background: Cutaneous leishmaniasis is a parasitic disease, which is difficult to treat due to high drug resistance and adverse side effects. Photodynamic therapy by ultraviolet radiation using materials with high photocatalytic features like titanium dioxide nanoparticles (TiO₂-NPs) is an emerging treatment for this disease. In this study, TiO₂-NPs with ultraviolet (UV) radiation were administered as photodynamic therapy against Leishmania Major (LM) promastigotes.Methods: Two forms of TiO₂ viz. including Anatase and Rutile were administered in two UV ranges< UVA and UVB for different time periods (30 and 60 min). Finally, 24 and 48 h after incubation, the MTS test was performed and cell survival percentage was calculated.Results: The mean size of Anatase and Rutile-NPs is approximately 32.5 and 50.9 nm respectively by DLS and FE-SEM, and crystal phase is emphasized by XRD. The combined treatment of LM with TiO₂-NPs and UV has significant effects on LM promastigotes, which vary depending on NP and UV types. The synergistic effect was anticipated in the groups irradiated by UV-B in the presence of Rutile NPs.Conclusion: The combined treatment with UV- radiation and TiO₂-NPs can be effective in killing the promastigotes of Leishmania major. The proper concentration of NPs and the type of UV-radiation must be taken into consideration. The results suggest improved treatment methods, after proper in vivo studies.     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

Low-Temperature Synthesis of Anatase TiO2 Nanoparticles with Tunable Surface Charges for Enhancing Photocatalytic Activity

In this work, the positively or negatively charged anatase TiO₂ nanoparticles were synthesized via a low temperature precipitation-peptization process (LTPPP) in the presence of poly(ethyleneimine) (PEI) and poly(sodium4- styrenesulfonate) (PSS). X-ray diffraction (XRD) pattern and high-resolution transmission electron microscope (HRTEM) confirmed the anatase crystalline phase. The charges of the prepared TiO₂, PEI-TiO₂ and PSS-TiO₂ nanoparticles were investigated by zeta potentials. The results showed that the zeta potentials of PEI-TiO₂ nanoparticles can be tuned from +39.47 mV to +95.46 mV, and that of PSS-TiO₂ nanoparticles can be adjusted from −56.63 mV to −119.32 mV. In comparison with TiO₂, PSS-TiO₂ exhibited dramatic adsorption and degradation of dye molecules, while the PEI modified TiO₂ nanoparticles showed lower photocatalytic activity. The photocatalytic performances of these charged nanoparticles were elucidated by the results of UV-vis diffuse reflectance spectra (DRS) and the photoluminescence (PL) spectra, which indicated that the PSS-TiO₂ nanoparticles showed a lower recombination rate of electron-hole pairs than TiO₂ and PEI-TiO₂.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.