Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.     

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.     

Direct electron transfer of Horseradish peroxidase on porous structure of screen-printed electrode

Disposable hydrogen peroxide biosensor was developed based on the direct electron transfer of horseradish peroxidase (HRP) on porous screen-printed carbon electrodes. Conventional screen-printing process was manually performed to fabricate the planar carbon electrodes, which were endowed with porous surfaces especially after anodizing pretreatment. The cyclic voltammetry experiment indicated a pair of stable and well-defined redox peaks with a formal potential of -0.33 V. And the formal potential was pH-dependent, having a slope of -55.2 mV/pH which indicated one electron transfer. The heterogeneous electron transfer rate constant k(s) was estimated to be 13.28+/-4.80s(-1). Additionally, the sensitivity was 143.3 mAM(-1)cm(-2) and the linear range was from 5.98 to 35.36 microM. In conclusion, the present work achieved the direct electron transfer of HRP on screen-printed electrodes without any promoters. The porous structure of screen-printed carbon electrodes facilitated the direct electron transfer between the active sites of HRP and the electrodes due to large amounts of conductive sites available on the surface for contacting with enzyme molecules. Moreover, the proposed biosensor could be mass-produced at low price, promising for commercial application.     

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.     

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.     

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.     

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV-Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H(2)O(2) in the linear range from 5×10(-6) to 1×10(-3) mol L(-1) with a detection limit of 2.1×10(-6) mol L(-1) (based on the S/N=3).     

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.     

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.     

Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films

Protein-chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about -0.33V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about -0.46V which was not stable. All these peaks are located at the potentials characteristic of heme Fe(III)/Fe(II) redox couples of the proteins. The electrochemical parameters such as formal potentials (E degrees (')) and apparent heterogeneous electron-transfer rate constants (k(s)) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein-CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.     

Direct electrochemistry and electrocatalysis based on a film of horseradish peroxidase intercalated into Ni-Al layered double hydroxide nanosheets

Positively charged Ni-Al layered double hydroxide nanosheets (Ni-Al LDHNS) have been used for the first time as matrices for immobilization of horseradish peroxidase (HRP) in order to fabricate enzyme electrodes for the purpose of studying direct electron transfer between the redox centers of proteins and underlying electrodes. X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) revealed that the HRP-Ni-Al LDHNS film had an ordered structure and that HRP was intercalated into Ni-Al LDHNS with a monolayer arrangement. Field emission scanning electron microscopy (FESEM) showed that the HRP-Ni-Al LDHNS film had a uniform, porous morphology. UV-vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the Ni-Al LDHNS film. The immobilized HRP in Ni-Al LDHNS on the surface of a glassy carbon electrode (GCE) exhibited good direct electrochemical and electrocatalytic responses to the reduction of hydrogen peroxide (H(2)O(2)) and trichloroacetic acid (TCA). The resulting H(2)O(2) biosensor showed a wide linear range from 6.00x10(-7)M to 1.92x10(-4)M, low detection limit (4.00x10(-7)M) and good stability. The results show that Ni-Al LDHNS provide a novel and efficient platform for the immobilization of enzymes and realizing direct electrochemistry and that the materials have potential applications in the fabrication of third-generation biosensors.     

Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

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.     

Electrochemistry and electrocatalysis of myoglobin immobilized in sulfonated graphene oxide and Nafion films

In this study, sulfonated graphene oxide (SGO) was synthesized and characterized by Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). It was used to make Mb–SGO–Nafion composite films by coating myoglobin (Mb) on the glassy carbon electrodes (GCE). Positions of the Soret absorption bands suggested that Mb retained its native conformation in the films. Mb–SGO–Nafion film modified electrode showed a pair of well-defined and nearly reversible cyclic voltammetry peaks at around −0.39 V versus saturated calomel electrode (SCE) in pH 7.0 buffers, characteristic of heme Fe(III)/Fe(II) redox couples. Electrochemical parameters such as electron transfer rate constant (k_s) and formal potential (E^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. Experimental data demonstrated that the electron transfer between Mb and electrode was greatly facilitated and showed good electrocatalytic properties toward various substrates, such as H₂O₂ and NaNO₂, with significant lowering of reduction overpotential.     

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.     

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.     

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV–Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.     

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.