Direct electron transfer between hemoglobin and pyrolytic graphite electrodes enhanced by Fe(3)O(4) nanoparticles in their layer-by-layer self-assembly films

Alternate adsorption of negatively charged Fe(3)O(4) nanoparticles from their pH 8.0 aqueous dispersions and positively charged hemoglobin (Hb) from its pH 5.5 buffers on solid substrates resulted in the assembly of {Fe(3)O(4)/Hb}(n) layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the film growth. A pair of well-defined, nearly reversible CV peaks for HbFe(III)/Fe(II) redox couples was observed for {Fe(3)O(4)/Hb}(n) films on pyrolytic graphite (PG) electrodes. Although the multilayered films grew linearly with the number of Fe(3)O(4)/Hb bilayers (n) and the amount of Hb adsorbed in each bilayer was generally the same, the electroactive Hb could only extend to 6 bilayers. This indicates that only those Hb molecules in the first few bilayers closest to the electrode surface are electroactive. The electrochemical parameters such as the apparent heterogeneous electron transfer rate constant (k(s)) were estimated by square wave voltammetry (SWV) and nonlinear regression. The Soret absorption band position of Hb in {Fe(3)O(4)/Hb}(6) films showed that Hb in the films retained its near native structure in the medium pH range. The {Fe(3)O(4)/Hb}(6) film electrodes also showed good biocatalytic activity toward reduction of oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite. The electrochemical reduction overpotentials of these substrates were lowered significantly by {Fe(3)O(4)/Hb}(n) films.     

Assembly of myoglobin layer-by-layer films with poly(propyleneimine) dendrimer-stabilized gold nanoparticles and its application in electrochemical biosensing

The small-sized Au nanoparticles (3 nm) were prepared by reduction of HAuCl(4) in the presence of poly(propyleneimine) (PPI) dendrimers, forming the stable PPI-Au nanoclusters in aqueous medium. The PPI-Au nanoclusters might take a kind of "core-shell" structure, in which several PPI molecules were attached on the surface of one gold nanoparticle. The PPI-Au nanoclusters in aqueous dispersions and myoglobin (Mb) in its buffers at pH 5.0 were then alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes and other solid substrates, forming {PPI-Au/Mb}(n) layer-by-layer films, which was confirmed by cyclic voltammetry (CV) and quartz crystal microbalance (QCM). {PPI-Au/Mb}(n) films on PG electrodes demonstrated a pair of well-defined and quasi-reversible CV reduction-oxidation peaks for Mb heme Fe(III)/Fe(II) couple and good electrocatalytic properties toward reduction of oxygen and hydrogen peroxide. Compared with {Au/Mb}(n) multilayer films containing no dendrimers and {PAMAM/Mb}(n) films assembled by polyamidoamine (PAMAM) dendrimers and Mb but in the absence of Au nanoparticles, {PPI-Au/Mb}(n) films showed better electrochemical behaviors and catalytic performances, which may be attributed to the unique structure of PPI-Au nanoclusters and good conductivity of gold nanoparticles. This novel kind of protein multilayer films assembled with dendrimer-stabilized gold nanoparticles may provide a new and general approach to fabricate the biosensors and bioreactors based on the direct electrochemistry of proteins or enzymes.     

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.     

Voltammetric studies of hemoglobin-coated polystyrene latex bead films on pyrolytic graphite electrodes

A novel hemoglobin (Hb)-coated polystyrene (PS) latex bead film was deposited on pyrolytic graphite (PG) electrode surface. In the first step, positively charged Hb molecules in pH 5.0 buffers were adsorbed on the surface of negatively charged, 500 nm diameter PS latex beads bearing sulfate groups by electrostatic interaction. The aqueous dispersion of Hb-coated PS particles was then deposited on the surface of PG electrodes and, after evaporation of the solvent, Hb-PS films were formed. The Hb-PS film electrodes exhibited a pair of well-defined, quasi-reversible cyclic voltammetric (CV) peaks at about −0.36 V vs. SCE in pH 7.0 buffers, characteristic of Hb heme Fe(III)/Fe(II) redox couples. Positions of Soret absorption band of Hb-PS films suggest that Hb retains its near-native structure in the films in its dry form and in solution at medium pH. The Hb in PS films was also acted as a catalyst to catalyze electrochemical reduction of various substrates such as trichloroacetic acid (TCA), nitrite, oxygen and hydrogen peroxide.     

Assembly of electroactive layer-by-layer films of hemoglobin and polycationic poly(diallyldimethylammonium)

Layer-by-layer (PDDA/Hb)(n) films were assembled by alternate adsorption of positively charged poly(diallyldimethylammonium) (PDDA) and negatively charged hemoglobin (Hb) at pH 9.2 from their aqueous solutions on pyrolytic graphite electrodes and other substrates. The assembly process was monitored and confirmed by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). CVs of (PDDA/Hb)(n) films showed a pair of well-defined, nearly reversible peaks at about -0.34 V vs SCE at pH 7.0, characteristic of Hb heme Fe(III)/Fe(II) redox couple. Positions of Soret absorption band and infrared amide II band of Hb in (PDDA/Hb)(8) films suggest that Hb in the films keeps its secondary structure similar to its native state. The electrochemical parameters of (PDDA/Hb)(8) films were estimated by square wave voltammetry, and the thickness of the PDDA/Hb bilayer was estimated by QCM and scanning electron microscopy. Trichloroacetic acid and nitrite (NO(2)(-)) were catalytically reduced at (PDDA/Hb)(8) film electrodes. The electrochemical catalytic reactions of O(2) and H(2)O(2) on (PDDA/Hb)(8) films were also studied.     

Self-assembly of electroactive layer-by-layer films of heme proteins with anionic surfactant dihexadecyl phosphate

Anionic surfactant dihexadecyl phosphate (DHP) with two hydrocarbon chains can be self-assembled into a double-layer structure with tail-to-tail configuration and negatively charged head groups toward outside in its aqueous dispersion. Due to this unique biomembrane-like structure, the "charge reversal" in DHP adsorption on solid surface was realized, and the DHP was successfully assembled with positively charged myoglobin (Mb) or hemoglobin (Hb) into {DHP/protein}(n) layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor or confirm the film assembly process. The {DHP/protein}(n) films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about -0.35 V vs SCE in pH 7.0 buffers, characteristic of the protein heme Fe(III)/Fe(II) redox couples. Based on the direct electrochemistry of heme proteins, the {DHP/protein}(n) films could also be used to electrochemically catalyze reduction of oxygen, hydrogen peroxide and nitrite with significant lowering of reduction overpotentials. Scanning electron microscopy (SEM), UV-vis spectroscopy, and reflectance absorption infrared (RAIR) spectroscopy were employed to characterize the {DHP/protein}(n) films, suggesting that the proteins in the films retain their near-native structure.     

Loading/release behavior of (chitosan/DNA)n layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage

In the present work, positively charged chitosan (CS) and negatively charged DNA were alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes, forming (CS/DNA)(n) layer-by-layer films. Cyclic voltammetry (CV) results showed that negatively charged electroactive probe, 9,10-anthraquinone-2,6-disulfonate (AQDS), could be loaded into the (CS/DNA)(n) films from its solution (1 mM at pH 7.0, containing 0.1 M NaCl), designated as (CS/DNA)(n)-AQDS, and then released from the films in blank buffers. The loading/release behavior of (CS/DNA)(n) films toward AQDS was found to be obviously different between double-stranded (dsDNA) and single-stranded DNA (ssDNA). The release rate of AQDS from (CS/dsDNA)(n) films was much slower than that from the ssDNA counterparts mainly because AQDS could be intercalated into the double helix structure of dsDNA despite the repulsion between likely charged AQDS and DNA. The loading/release behavior of (CS/DNA)(n) films toward AQDS in recognition of dsDNA and ssDNA was then successfully applied to electrochemically detect the damage of natural DNA caused by Fenton reaction. To further understand the essence of the interactions involved in the AQDS loading/release process for (CS/DNA)(n) films, comparison experiments were performed, in which either positively charged intercalator brilliant cresyl blue (BCB) was used to replace AQDS as the redox probe, or poly(diallyldimethylammonium) (PDDA) with relatively high positive charge density was used to replace CS as the constituent of layer-by-layer films with DNA. The loading/release behavior of DNA films toward electroactive intercalator may open new possibilities for dsDNA/ssDNA recognition and of DNA damage detection by electrochemistry.     

Electrostatic adsorption of heme proteins alternated with polyamidoamine dendrimers for layer-by-layer assembly of electroactive films

A novel thin film of heme proteins, including hemoglobin (Hb), myoglobin (Mb), and catalase (Cat), was successfully assembled layer by layer with polyamidoamine (PAMAM) dendrimers on different solid surfaces. At pH 7.0, protonated PAMAM possesses positive surface charges, whereas the proteins have net negative surface charges at pH above their isoelectric points. Thus, layer-by-layer {PAMAM/protein}(n)() films were assembled with alternate adsorption of oppositely charged PAMAM and proteins from their aqueous solutions mainly by electrostatic interaction. The assembly process was monitored by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). The growth of the protein multilayer films was regular and linear, whereas the electroactivity of the films was only extended to a few bilayers. CVs of {PAMAM/protein}(n)() films showed a pair of well-defined and nearly reversible peaks characteristic of the protein heme Fe(III)/Fe(II) redox couples. Although {PAMAM/Hb}(n)() and {PAMAM/Mb}(n)() films showed very similar properties, {PAMAM/Cat}(n)() films displayed different and unique characters. The substrates with biological or environmental significance, such as oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite, were catalytically reduced at {PAMAM/protein}(n)() film electrodes, showing the potential applicability of the films as new types of biosensors or bioreactors based on direct electrochemistry of the proteins. Both the electrochemical and electrocatalytic activity of {PAMAM/protein}(n)() films can be tailored precisely by controlling the number of bilayers or the film thickness.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes. Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about -0.34 V vs. saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of -0.47 V. The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (k(s)) and formal potentials (E (degrees ')) were estimated by square wave voltammetry with nonlinear regression analysis. UV-vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.     

Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes. Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about −0.34 V vs. saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of −0.47 V. The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (k_s) and formal potentials (E°′) were estimated by square wave voltammetry with nonlinear regression analysis. UV-vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.     

An amperometric hydrogen peroxide sensor based on immobilization of hemoglobin in poly(o-aminophenol) film at iron-cobalt hexacyanoferrate-modified gold electrode

A series of hybrid iron-cobalt hexacyanoferrate (FeCoHCF) films were electrodeposited on gold electrodes from solutions containing 6mM Fe(CN)(6)(3-) with different concentrations of Co(2+) and Fe(3+). FeCoHCF films deposited from solutions with different molar ratios of iron were studied by cyclic voltammetry, and their solid states were characterized by Fourier transform infrared spectroscopy. The kind of FeCoHCF film that deposited from a solution with a molar ratio of iron of 0.4 showed the largest response current to H(2)O(2) and was characterized by energy-dispersive X-ray spectroscopy. Therefore, the optimized FeCoHCF film was combined with nonconducting poly(o-aminophenol) (POAP) film that entrapped the hemoglobin (Hb) to construct hydrogen peroxide biosensor. The response current of the Hb/POAP/FeCoHCF/Au electrode (29.8 nA) was nearly 40 and was 1.5 times that of the Hb/POAP/Au (0.7 nA) and POAP/FeCoHCF/Au (20 nA) electrodes, respectively. The Michaelis-Menten constant of Hb in the Hb/POAP/FeCoHCF/Au film was 9.31 mM. These results show that the immobilized Hb in the Hb/POAP/FeCoHCF/Au film exhibits higher catalytic activity and larger response current to H(2)O(2) by the mediation of FeCoHCF. In addition, effects of applied potential, solution pH, and electroactive interferent on the response current of the Hb/POAP/FeCoHCF/Au electrode were investigated in detail.     

Amphiphilic core-shell nanoparticles with poly(ethylenimine) shells as potential gene delivery carriers

Spherical, well-defined core-shell nanoparticles that consist of poly(methyl methacrylate) (PMMA) cores and branched poly(ethylenimine) shells (PEI) were synthesized via a graft copolymerization of methyl methacrylate from branched PEI induced by a small amount of tert-butyl hydroperoxide. The PMMA-PEI core-shell nanoparticles were between 130 to170 nm in diameter and displayed zeta-potentials near +40 mV at pH 7 in 1 mM aqueous NaCl. Plasmid DNA (pDNA) was mixed with nanoparticles and formed complexes of approximately 120 nm in diameter and was highly monodispersed. The complexes were characterized with respect to their particle size, zeta-potential, surface morphology, and DNA integrity. The complexing ability of the nanoparticles was strongly dependent on the molecular weight of the PEI and the thickness of the PEI shells. The stability of the complexes was influenced by the loading ratio of the pDNA and the nanoparticles. The condensed pDNA in the complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxity studies using MTT colorimetric assays suggested that the PMMA-PEI (25 kDa) core-shell nanoparticles were three times less toxic than the branched PEI (25 kDa). Their transfection efficiencies were also significantly higher. Thus, the PEI-based core-shell nanoparticles show considerable potential as carriers for gene delivery.     

An electrochemical investigation of hemoglobin and catalase incorporated in collagen films

Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.     

Direct electrochemistry of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV–Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb–HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

Direct electrochemistry of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV-Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb-HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

Protein-filled polyelectrolyte microcapsules in the design of enzymic microdiagnostics

Encapsulation of enzymes (lactate dehydrogenase and urease) in polyelectrolyte shells was assessed with a view to designing enzymic microdiagnostics for low-molecular compounds in native biological fluids. Polyelectrolyte microcapsules were prepared with two polyanions [poly(styrenesulfonate) PSS and dextran sulfate DS] and two polycations [poly(allylamine) PAA and poly(diallyldimethylammonium) PDADMA]; calcium carbonate microspherulites with embedded enzymes served as “cores.” It was demonstrated that the main problem in making such a biosensor is to select a pair of oppositely charged polyelectrolytes that would be optimal for enzyme functioning. The best pairs were PAA/DS and PAA/PSS for lactate dehydrogenase, and PSS/PAA and PSS/PDADMA for urease. We designed and prepared enzyme-containing microcapsules differing in polyelectrolyte composition and number of layers, and investigated their properties.     

Electrochemical fabrication and evaluation of highly sensitive nanorod-modified electrodes for a biotin/avidin system

Bioaffinity sensors need to be rapid, specific, and highly sensitive. To realize these features, electrodes that can elicit high electrochemical performance are necessary. In this study, we developed nanorod array electrode and performed cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments to study the interfacial properties of the nanorod array electrode with Fe(CN)(6)(3-/4-) as the redox molecules. Results showed that both the CV and EIS measurements captured very well the resistive and capacitive changes due to the adsorption of functionalizing molecules and the coupling between avidin and biotin. The EIS measurements were more sensitive in discriminating small changes caused by the surface adsorption of various molecules. The use of avidin-functionalized gold nanorod modified electrodes had led to much increased detection sensitivity along with a detection-limit as low as 1 ng/mL of biotin.     

Facile synthesis of Fe(3)O(4)@Al(2)O(3) core-shell nanoparticles and their application to the highly specific capture of heme proteins for direct electrochemistry

In this study, magnetic core-shell Fe(3)O(4)@Al(2)O(3) nanoparticles (NPs) attached to the surface of a magnetic glassy carbon electrode (MGCE) were used as a functional interface to immobilize several heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) for fabricating protein/Fe(3)O(4)@Al(2)O(3) film. Transmission electron microscope, UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the films. With the advantages of the magnetism and the excellent biocompatibility of the Fe(3)O(4)@Al(2)O(3) NPs, the protein/Fe(3)O(4)@Al(2)O(3) film could be easily fabricated in the present of external magnetic field, and well retained the bioactivity of the immobilized proteins, hence dramatically facilitated direct electron transfer of heme proteins and excellent electrocatalytic behaviors towards H(2)O(2) were demonstrated. The presented system avoids the complex synthesis for protecting Fe(3)O(4) NPs, supplies a facile, low cost and universal way to immobilize proteins, and is promising for construction of third-generation biosensors and other bio-magnetic induction devices.