A peptide cross-linked polyacrylamide hydrogel for the detection of human neutrophil elastase

Polyacrylamide hydrogel films cross-linked with the peptide sequence AAPVAAK were synthesised on piezoelectric quartz crystals. Degradation of the hydrogel films was monitored using a combination of quartz crystal microbalance (QCM) and electrochemical impedance measurements. The films were shown to degrade in the presence of human neutrophil elastase (HNE). The rate of degradation was directly related to the enzyme activity making the system suitable for the detection of HNE in the activity range from 0.72 to 30 U mL⁻¹. The films were not affected by non-specific adsorption. Film degradation was only accompanied by changes in the QCM signal, while no significant impedance change was observed during degradation.     

EQCN based cholinesterase biosensors

The binding of acetylcholinesterase (AChE) to a propidium-modified piezoelectric quartz crystal and its surface enzymatic activity have been investigated.Propidium binds to a site remote to the active center of AChE - the peripheral anionic site (PAS) - which is located on the rim of the gorge to the active site.The gold electrodes of the quartz crystal were first modified with 11-mercaptoundecanoic acid to which propidium was coupled. AChE binding was monitored by a quartz crystal nanobalance (QCN), followed by amperometric activity evaluation of the AChE loaded on the sensor. Interestingly, the binding is strong but does not inhibit AChE. However, an excess of propidium in solution inhibits the immobilized enzyme. The surface enzymatic activities observed depend on the amount of enzyme and differ according to the type and species, i.e. number of enzyme subunits (Electrophorus electricus tetrameric, Drosophila melanogaster mono- and dimeric form - DmAChE).The operational stability and regeneration, effect of propidium in solution and detection limit for substrate for various AChEs were investigated amperometrically.     

A voltammetric and nanogravimetric study of Te underpotential deposition on Pt in perchloric acid medium

This work describes the study of Te underpotential deposition on Pt in acid media using cyclic voltammetry, rotating ring-disc electrode and electrochemical quartz crystal microbalance techniques. The voltammetric results indicate the presence of two dissolution peaks in the positive scan with a total charge density of 420 μC cm⁻². These phenomena are attributed to the deposition of one Te monolayer with the occupancy of two active Pt sites by each ad-atom. This is confirmed by rotating ring-disc electrode results. The electrochemical quartz crystal microbalance (EQCM) experiments yielded the small mass variation of −32 ng cm⁻² (while the theoretical one is −140.4 ng cm⁻² for a complete Te monolayer). This low value can be attributed to the simultaneous adsorption of water, perchlorate anions and the formation of platinum oxide.     

Development of highly sensitive non-enzymatic sensor for the selective determination of glucose and fabrication of a working model

Copper oxide (CuO)/copper oxalate (CuOx) modified non-enzymatic electrochemical sensor for the detection of glucose in alkaline medium was fabricated by electrochemical anodisation of copper electrodes in potassium oxalate solution. Morphology of the modified copper electrode was studied by Scanning Electron Microscopy (SEM) and its electrochemical behaviour by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). The formation of CuOx on the copper electrode was confirmed by the Infra-red Reflection Absorption Spectrum (IRRAS). The modified electrodes were found to be microporous and rough. Linear Sweep Voltammetry (LSV) and amperometry were adopted to investigate the direct electrocatalytic oxidation of glucose on CuO/CuOx modified electrode in alkaline medium which showed excellent catalytic activity. The best performance of the sensor was obtained at 0.7 V and in 0.1 M sodium hydroxide (NaOH). At this optimum potential, the sensor was highly selective to glucose in the presence of ascorbic acid (AA) and uric acid (UA) which are common interfering species in biological fluids. The sensitivity was found to be very high (1890 μA mM⁻¹ cm⁻²) with excellent linearity (R = 0.9999) up to 15 mM having a low detection limit of 0.05 μM (S/N = 3). The modified electrode was tested for glucose level in blood serum. Based on the optimised conditions, a working model of the sensor was made and successfully tested for glucose.     

Immobilization, direct electrochemistry and electrocatalysis of hemoglobin on colloidal silver nanoparticles-chitosan film

This paper reports on the fabrication and characterization of hemoglobin (Hb)-colloidal silver nanoparticles (CSNs)-chitosan film on the glassy carbon electrode and its application on electrochemical biosensing. CSNs could greatly enhance the electron transfer reactivity of Hb as a bridge. In the phosphate buffer solution with pH value of 7.0, Hb showed a pair of well-defined redox peaks with the formal potential (E^0′) of −0.325 V (vs. SCE). The immobilized Hb in the film maintained its biological activity, showing a surface-controlled process with the heterogeneous electron transfer rate constant (k_s) of 1.83 s⁻¹ and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide (H₂O₂). The linear range for the determination of H₂O₂ was from 0.75 μM to 0.216 mM with a detection limit of 0.5 μM (S/N = 3). Such a simple assemble method could offer a promising platform for further study on the direct electrochemistry of other redox proteins and the development of the third-generation electrochemical biosensors.     

Direct electron transfer of hemoglobin in a CdS nanorods and Nafion composite film on carbon ionic liquid electrode

In this paper the direct electron transfer of hemoglobin (Hb) was carefully investigated by using a room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) modified carbon paste electrode (CILE) as the basal working electrode. Hb was immobilized on the surface of CILE with the nanocomposite film composed of Nafion and CdS nanorods by a step-by-step method. UV–vis and FT-IR spectra showed that Hb in the composite film remained its native structure. The direct electrochemical behaviors of Hb in the composite film were further studied in a pH 7.0 phosphate buffer solution (PBS). A pair of well-defined and quasi-reversible cyclic voltammetric peaks of Hb was obtained with the formal potential (E⁰′) at −0.295 V (vs. SCE), which was the characteristic of heme Fe(III)/Fe(II) redox couples. The direct electrochemistry of Hb was achieved on the modified electrode and the apparent heterogeneous electron transfer rate constant (k_s) was calculated to be 0.291 s⁻¹. The formal potentials of Hb Fe(III)/Fe(II) couple shifted negatively with the increase of buffer pH and a slope value of −45.1 mV/pH was got, which indicated that one electron transfer accompanied with one proton transportation. The fabricated Hb sensor showed good electrocatalytic manner to the reduction of trichloroacetic acid (TCA).     

Direct electron transfer of hemoglobin immobilized in a mesocellular siliceous foams supported room temperature ionic liquid matrix and the electrocatalytic reduction of H2O2

Room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM·PF₆) has been successfully immobilized on mesocellular siliceous foams (MSFs) by using a specific annealing method. Nitrogen adsorption/desorption isotherms and scanning electron microscopy (SEM) images reveal that most pores of MSFs are filled with the RTIL and the outer surfaces of MSFs are covered with the RTIL. When hemoglobin (Hb) is immobilized with the resulting hybrid material on a glassy carbon electrode (GCE), a pair of well-defined and quasi-reversible voltammetric peaks for Hb Fe(III)/Fe(II) is obtained. Its formal potential is −0.330 V (vs. saturated calomel electrode) in pH 7.0 phosphate buffer solution (PBS). The peak currents are much larger than those of Hb immobilized with MSFs or BMIM·PF₆-MSFs mixture. This indicates that the hybrid material has stronger promotion to the direct electron transfer of Hb, which is related to the effective immobilization of BMIM·PF₆ on MSFs. The electron-transfer rate constant (k_s) is estimated to be 1.91 s⁻¹. The immobilized Hb retains its native conformation and shows high electrocatalysis to the reduction of H₂O₂. Under the optimized experimental conditions, the catalytic current is linear to the concentration of H₂O₂ from 0.2 to 28 μM, and the detection limit is 8 × 10⁻⁸ M (S/N = 3). The linear range is wider than those for Hb immobilized with MSFs or BMIM·PF₆-MSFs mixture. Thus, the MSFs supported RTILs hybrid material is an ideal matrix for protein immobilization and biosensor fabrication.     

Assessment on the use of biodiesel in cold weather: Pour point determination using a piezoelectric quartz crystal

In order to use biodiesel safely, as an alternative fuel for diesel engines, without fear of cold weather, the pour point of the blends needs to be estimated. This paper is aimed to propose an alternative and easy to use methodology, based on a piezoelectric quartz crystal, to determine the pour point of biodiesels and blended fuels.Impedance and phase of impedance vs. frequency of the piezoelectric quartz crystal change significantly during cooling of biodiesel and biodiesel blended fuels and allows to confirm the role of ethanol as a cold flow improver for biodiesel. Pour point is readily determined by finding the minimum series or parallel frequencies of a barred piezoelectric quartz crystal in contact with the biodiesel blended fuel along cooling. This new methodology only needs the measurement of series frequency, which can be accomplished with high precision by connecting a frequencymeter to a home made oscillator that drives the piezoelectric quartz crystal. Although inexpensive, this new methodology is no more based on visual inspection as the ASTM D97 method, and allows data to be acquired more frequently than the 3 °C intervals recommended by the time consuming standard methodology. In the new proposed methodology, data is acquired while the fuel is at the controlled temperature, which is not possible with the ASTM method, where the test jar needs to be removed from the thermostatic bath for visual inspection.Pour points of biodiesel blends with a commercial diesel fuel determined by this new methodology were compared with the ones obtained by the official ASTM methodology. For samples with pour points ranging from 2.3 °C (pure biodiesel) to −15.0 °C (pure commercial fuel diesel), median pour point values obtained for replicate measurements performed by the two methodologies were not statistically different (α = 0.05), although the results obtained by the new methodology were more precise.     

Direct electron-transfer and electrochemical catalysis of hemoglobin immobilized on mesoporous Al2O3

The direct electrochemistry of hemoglobin (Hb) has been achieved by immobilizing Hb on mesoporous Al₂O₃ (meso-Al₂O₃) film modified glassy carbon (GC) electrode. Meso-Al₂O₃ shows significant promotion to the direct electron-transfer of Hb, thus it exhibits a pair of well defined and quasi-reversible peaks with a formal potential of −0.345 V (vs. SCE). The electron-transfer rate constant (k_s) is estimated to be 3.17 s⁻¹. The immobilized Hb retains its biological activity well and shows high catalytic activity to the reduction of hydrogen peroxide (H₂O₂) and nitrite (NO₂⁻). Under the optimized experimental conditions, the catalytic currents are linear to the concentrations of H₂O₂ and NO₂⁻ in the ranges of 0.195-20.5 μM and 0.2-10 mM, respectively. The corresponding detection limits are 1.95 × 10⁻⁸ M and 3 × 10⁻⁵ M (S/N = 3). The resulting protein electrode has high thermal stability and good reproducibility due to the protection effect of meso-Al₂O₃. Ultraviolet visible (UV-vis) absorption spectra and reflection-absorption infrared (RAIR) spectra display that Hb keeps almost natural structure in the meso-Al₂O₃ film. The N₂ adsorption-desorption experiments show that the pore size of meso-Al₂O₃ is about 14.4 nm, suiting for the encapsulation of Hb (average size: 5.5 nm) well. Therefore, meso-Al₂O₃ is an alternative matrix for protein immobilization and biosensor preparation.     

Hemoglobin immobilized on whisker-like carbon composites and its direct electrochemistry

We developed a novel mesoporous carbon/whisker-like carbon (MCWC) composite which can promote the direct electron transfer of hemoglobin (Hb) immobilized on its surface. The cyclic voltammetric results showed that Hb immobilized on the surface of the MCWC composite could undergo a direct quasi-reversible electrochemical reaction. Its formal redox potential, E^0′ is −0.313 V in the phosphate buffer solution (pH 6.9) at a scan rate of 200 mV/s and is almost independent of the scan rate in the range of 100-600 mV/s. The dependence of E^0′ on the pH of phosphate buffer solution indicated that the redox reaction of Hb includes a one-electron-transfer reaction process coupled with one-proton-transfer. The experiment obtained larger value of electron transfer rate constant, k_s, than that of Hb immobilized on other carriers reported previously due to its special structure of loosely packed nanometer-scale carbon whiskers and thus formed “V-type” nano-pores. Furthermore, Hb immobilized on the surface of the MCWC composite can retain the stable bioelectrocatalytic activity for the reduction of H₂O₂.     

A DNA electrochemical sensor with poly-l-lysine/single-walled carbon nanotubes films and its application for the highly sensitive EIS detection of PAT gene fragment and PCR amplification of NOS gene

A sensitive and novel DNA electrochemical biosensor for the detection of the transgenic plants gene fragment by electrochemical impedance spectroscopy (EIS) was presented. The well-dispersed carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) were dripped onto the carbon paste electrode (CPE) surface firstly, and poly-l-lysine films (pLys) were subsequently electropolymerized by cyclic voltammetry (CV) to prepare pLys/SWNTs/CPE. The morphology of pLys/SWNTs films was examined using a field emission scanning electron microscope (SEM). The pLys/SWNTs films modified electrode exhibited very good conductivity. DNA probes were easily immobilized on the poly-l-lysine films via electrostatic adsorption. The hybridization events were monitored with electrochemical impedance spectroscopy using [Fe(CN)₆]^3−/4− as indicator. The PAT gene fragment from phosphinothricin acetyltransferase gene was detected by this DNA electrochemical sensor. The dynamic detection range of this sensor to the PAT gene fragment was from 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ mol/L. A detection limit of 3.1 × 10⁻¹³ mol/L could be estimated. The PCR amplification of NOS gene from the sample of a kind of transgenic modified bean was also detected satisfactorily by EIS.     

Layered double hydroxides functionalized with anionic surfactant: Direct electrochemistry and electrocatalysis of hemoglobin

Direct electrochemistry of hemoglobin (Hb), which was immobilized on the glass carbon electrode (GCE) modified with Zn-Al layered double hydroxide (LDH) functionalized with sodium dodecylsulfonate (SDS), was investigated. The resulting electrode (Hb/LDH-SDS/GCE) gave a well-defined redox couple for HbFe(III)/Fe(II) with a formal potential of about −0.34 V (vs. AgCl/Ag) in pH 7.0 buffer. The electron-transfer rate constant was estimated to be 2.6 s⁻¹. The Hb/LDH-SDS/GCE exhibited a remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H₂O₂). The low calculated apparent Michaelis-Menten constant () was 456 μM. Based on the high catalytic activity of Hb immobilized on LDH-SDS modified electrode to the reduction of H₂O₂, LDH functionalized with SDS is expected to have widely potential applications for development of new biosensors and biocatalysis.     

Filling behavior of ZnO nanoparticles into opal template via electrophoretic deposition and the fabrication of inverse opal

Combining colloidal crystal template (artificial opal) and electrophoretic deposition (EPD) process, well-ordered ZnO inverse opal can be formed by finding the optimum driving potential of EPD. Through providing the various driving potentials from −25 V, −10 V, −5 V to −2.5 V, the different mechanism of electrophoretically depositing ZnO nanoparticles into the colloidal crystal template was determined by the SEM observation of the filled templates. Because the nano-channels of colloidal crystal template are the network type, the results of surface jam, incomplete filling and perfect filling are found under specific applied voltages. The high-quality ZnO inverse opal can be only fabricated under the perfect nano-channel-filling condition. The filling behavior can be monitored dynamically by tracing the current transients, and the optimum conditions for filling the interstitial spaces of templates constructed from colloidal particles with 180 nm and 300 nm diameter can be obtained by applying a voltage of −5 V and −15 V, respectively. After the complete filling of ZnO nanoparticles into the colloidal crystal template consisting of 300 nm colloids, high-quality ZnO photonic crystal possessing an absorptive peak at the wavelength of 560 nm can be fabricated by removing the template. It is expected that the EPD can find extensive applications for preparing photonic crystals of various oxides only if their nanoparticles are available.     

Self-assembled monolayers (SAMs) of cobalt tetracarboxylic acidchloride phthalocyanine covalently attached onto a preformed mercaptoethanol SAM: A novel method

A feasible method of fabricating phthalocyanine sensor was developed by covalent attachment of cobalt tetracarboxylic acidchloride phthalocyanine (CoTCACIPc) onto a preformed 2-mercaptoethanol (2-ME) self-assembled monolayer (SAM) modified gold electrode (designated as CoTCACIPc-2-ME-SAM). The surface concentration of the CoTCACIPc was found to be 4.58 × 10⁻¹⁰ mol/cm². The sensor gave a linear response to l-cysteine over the concentration range 0.28-20 μM with a detection limit of 5 × 10⁻⁷ M and best response time of 2 s.     

Electrochemistry and electrocatalysis of hemoglobin on multi-walled carbon nanotubes modified carbon ionic liquid electrode with hydrophilic EMIMBF4 as modifier

The direct electrochemistry of hemoglobin (Hb) on multi-walled carbon nanotubes (MWCNTs) modified carbon ionic liquid electrode (CILE) was achieved in this paper. By using a hydrophilic ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF₄) as the modifier, a new CILE was fabricated and further modified with MWCNTs to get the MWCNTs/CILE. Hb molecules were immobilized on the surface of MWCNTs/CILE with polyvinyl alcohol (PVA) film by a step-by-step method and the modified electrode was denoted as PVA/Hb/MWCNTs/CILE. UV-vis and FT-IR spectra indicated that Hb remained its native structure in the composite film. Cyclic voltammogram of PVA/Hb/MWCNTs/CILE showed a pair of well-defined and quasi-reversible redox peaks with the formal potential (E^0′) of −0.370 V (vs. SCE) in 0.1 mol/L pH 7.0 phosphate buffer solution (PBS), which was the characteristic of the Hb heme Fe^III/Fe^II redox couples. The redox peak currents increased linearly with the scan rate, indicating the direct electron transfer was a surface-controlled process. The electrochemical parameters of Hb in the film were calculated with the results of the electron transfer coefficient (α) and the apparent heterogeneous electron transfer rate constant (k_s) as 0.49 and 1.054 s⁻¹, respectively. The immobilized Hb in the PVA/MWCNTs composite film modified CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA) and hydrogen peroxide. So the proposed electrode showed the potential application in the third generation reagentless biosensor.     

Oxygen binding and other physical properties of human hemoglobin made in yeast

Wagenbach et al. (1991, BioTechnology, 9, 57–61) haverecently developed a system for producing soluble recombinanttetrameric hemoglobin in yeast: hemoglobin begins to appear4– 5 h after induction with galactose,- and ß-globinchains fold in vivo and endogeneously produced heme is incorporatedinto hemoglobin tetramers. We have further characterized theoxygen-binding properties, as well as the tetramer stability,of recombinant human Hb A made in yeast. After purificationby ion-exchange chromatography, a single band at the same positionas normal human Hb A was obtained using cellulose acetate electrophoresis.Although the oxy and deoxy forms of purified recombinant HbA made in yeast were spectrophotometrically identical to nativehuman Hb A, the oxygen-binding curve was shifted slightly leftof that for native human Hb A. Further purification of recombinanthemoglobin by FPLC revealed two fractions: one (fraction B)with low cooperativity and high oxygen affinity, and the other(fraction A) with almost identical cooperativity and oxygenaffinity compared with native human Hb A. The Bohr effect offraction A was also identical to native human Hb A. Hemoglobinin fraction B with lowered cooperativity precipitated -1.5 timesfaster than normal human Hb A during mechanical agitation, whilehemoglobin in fraction A with normal cooperativity precipitatedwith kinetics identical to native human Hb A. These resultssuggest that some of the recombinant molecules made in yeastfold improperly, and that these molecules may exhibit decreasedcooperativity for oxygen binding and decreased stability. Thissystem should now allow the evaluation of the folding differenceswhich promote hemoglobin function, and should also provide anefficient system which will facilitate the production of varioushemoglobin mutants for studying biochemical and biophysicalproperties of hemoglobin.     

Utilization of muscovite granite waste in the manufacture of ceramic tiles

Granite waste is by-product from a decorative rock industry. The present study aims to investigate the effect of muscovite granite waste on the physico-mechanical properties of ceramic tiles to demonstrate its suitability for industrial production. A series of flooring- and facing ceramic tiles were prepared by adding 20, 25, and 30 wt% muscovite granite waste into the batch compositions. The sintering behavior and degrees of densification of two kinds of ceramic tiles were evaluated by determining their physico-mechanical properties and characterizing them by scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. As expected, the facing ceramic tile (FacCT1) sample, containing wollastonite, hematite, anorthite, quartz, and cristobalite, with 20 wt% muscovite granite waste showed lower physico-mechanical properties than those of the flooring ceramic tile (FloCT3) sample, containing mullite, calcium aluminosilicate, quartz, and cristobalite, with 30 wt% muscovite granite waste. The reason is that lower firing temperatures cannot accelerate a complete fusion of the granite waste which behaves like an inert non-plastic material similar to quartz. These results illustrate the prospects of utilizing muscovite granite waste in ceramic tile production.     

A hydrogen peroxide sensor based on the peroxidase activity of hemoglobin immobilized on gold nanoparticles-modified ITO electrode

A novel ITO electrode surface modified with spherical and rod-shaped gold nanoparticles was prepared by a surfactant-assisted seeding growth approach, which provided a biocompatible matrix for the immobilization of hemoglobin (Hb). By electrochemical impedance measurements, gold nanoparticles modification and Hb immobilization on the electrode surfaces were characterized using [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ redox probe. Owing to the promoted electron transfer of Hb by gold nanoparticles, the Hb immobilized gold nanoparticles-modified ITO (Hb/Au/ITO) electrode exhibited an effective catalytic response to the reduction of H₂O₂ with good reproducibility and stability. The linear relationship existed between the catalytic current and the H₂O₂ concentration in the range of 1 × 10⁻⁵ to 7 × 10⁻³ M. The detection limit (S/N = 3) was 4.5 × 10⁻⁶ M.     

Ferroceneboronic acid for the electrochemical probing of interactions involving sugars

Ferroceneboronic acid (FcBA) was used as a redox-active probe suitable for monitoring of diol–boronate interactions. Voltammetric and amperometric measurements allowed to detect FcBA forms – free and bound in the boronate complex. In this way, the complexation interaction was studied for a set of saccharide molecules as model diols and the corresponding affinity equilibrium constants were determined. A shift of the peak potential on voltammograms accompanying formation of the boronate complex with FcBA was proposed as a probe for electrochemical characterization of surface-confined diol-containing structures. The model experiments were carried out using sorbitol- and 1,6-hexandiol-modified polyepichlorhydrin conjugates deposited on the electrodes; the former compound was able to form the boronate complex while no change of the peak potential for the latter conjugate was observed. This approach seems promising for artificial bioelectronic affinity receptors and technology of reagentless biosensors where the binding interaction directly stimulates a measurable electrochemical event.     

Application of the electrochemical quartz crystal microbalance technique to copper sonoelectrochemistry: Part 1. Sulfate-based electrolytes

The applicability of the electrochemical quartz crystal microbalance technique in an ultrasonic field created by an ultrasound probe is demonstrated for the electrodeposition of copper. Cyclic voltammetry and potentiostatic depositions in acidic sulfate-based copper electrolytes were performed at different ultrasonic intensities. The electrochemical quartz crystal microbalance was operated in ultrasonic fields with intensities up to 30 W cm⁻². For cyclic voltammetry, potential resolved and averaged (apparent) current efficiencies were calculated from mass and charge data in function of the amplitude of the ultrasonic horn. Ultrasound slightly affected the current efficiencies during copper deposition in cyclic voltammetry, but did not change the efficiencies during dissolution. During potentiostatic depositions the current efficiency increased from 84% to almost 100% upon application of ultrasound. Morphology of deposits prepared by potentiostatic depositions was analyzed by scanning electron microscopy, and found to be different at high ultrasonic intensities.