The electrochemical properties of thermophilic cytochrome P450 CYP119A2 at extremely high temperatures in poly(ethylene oxide)

The electrochemical measurements were carried out by using thermophilic cytochrome P450 CYP119A2 (P450st) modified with poly(ethylene oxide) (PEO) in PEO₂₀₀ as an electrochemical solvent. The PEO modified P450st gave clear reduction–oxidation peaks by cyclic voltammetry in oxygen-free PEO₂₀₀ up to 120 °C. The midpoint potential measured for the P450st was −120 mV vs. [Fe(CN)₆]⁴⁻/[Fe(CN)₆]³⁻ at 120 °C. The peak separation, ΔE, was 16 mV at 100 mV/s. The estimated electron transfer rate of PEO-P450st at 120 °C was 35.1 s⁻¹. The faster electron transfer reaction was achieved at higher temperatures. The electrochemical reduction of dioxygen was observed at 115 °C with the PEO-modified P450st system.     

Enhanced electrochemiluminescence based on Ru(bpy)32+-doped silica nanoparticles and graphene composite for analysis of melamine in milk

A sensitive electrochemiluminescence (ECL) sensor for melamine analysis was fabricated based on Ru(bpy)₃²⁺-doped silica (Ru(bpy)₃²⁺@SiO₂) nanoparticles and graphene composite. Spherical Ru(bpy)₃²⁺@SiO₂ nanoparticles with uniform size about 55 nm were prepared by the reverse microemulsion method. Since per Ru(bpy)₃²⁺@SiO₂ nanoparticle encapsulated a great deal of Ru(bpy)₃²⁺, the ECL intensity has been greatly enhanced, which resulted in high sensitivity. Due to its extraordinary electric conductivity, graphene improved the conductivity and accelerated the electron transfer rate. In addition, graphene could work as electronic channel improving the efficient luminophor amount participating in the ECL reaction, which further enhanced the ECL signal. This proposed sensor was used to melamine analysis and the ECL intensity was proportional to logarithmic melamine concentration range from 1 × 10⁻¹³ M to 1 × 10⁻⁸ M with the detect limit as low as 1 × 10⁻¹³ M. In application to detect melamine in milk, satisfactory recoveries could be obtained, which indicated this sensor having potential application in melamine analysis in real samples.     

Rapid formation of well-ordered self-assembled monolayers of dodecanethiol on polycrystalline gold by microwave irradiation

This article reports a rapid method of preparing self-assembled monolayers of dodecanethiol (C₁₂SH-SAMs) on polycrystalline gold by microwave irradiation (MWI, 650 W, duty cycle is 10%). The qualities of C₁₂SH-SAMs were characterized by both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results show that the C₁₂SH-SAMs formed by MWI in 120 s (C₁₂SH-SAMs_MWI,120 s) have low ionic permeability (the differential capacitance C_d values are independent of the scan rate and phase angle at 1 Hz Φ_1 Hz = 89 ± 0.9°), excellent electrochemical blocking ability towards the redox probe (the current i_MWI,120 s obtained from CV is lowest when compared to other SAMs and charge transfer resistance R_ct = (1.15 ± 0.19) × 10⁶ Ω cm²), and high surface coverage (99.996 ± 0.001%).     

Mediated electron transfer with P450cin

P450cin stereoselectively hydroxylated its natural substrate 1,8-cineole to 2β-hydroxy-1,8-cineole in an electrochemical cell which allowed for substitution of the natural cofactor NADPH by artificial redox mediators. Cobalt sepulchrate, phenosafranine, safranine T, FAD and FMN enabled artificial electron transfer from the platinum electrode to P450cin via the redox partner protein cindoxin. The highest product formation, 6.50 ± 0.60 nmol (product) nmol (P450)^?1 min^?1 cm^?2, was achieved using cobalt sepulchrate. Surprisingly, phenosafranine and safranine T enabled electron transfer even in the absence of NADPH, cindoxin, and cindoxin reductase, thereby illustrating that none of the natural redox partners is needed for product formation.     

Synthesis,characterization, and study of the photophysics and photocatalytic properties of the photoinitiated electron collector [{(phen)2Ru(dpp)}2RhBr2](PF6)5

The heterometallic photoinitiated electron collector [{(phen)₂Ru(dpp)}₂RhBr₂](PF₆)₅ (phen = 1,10-phenanthroline, dpp = 2,3-bis(2-pyridyl)pyrazine) has been synthesized and studied by spectroscopic, photophysical, electrochemical, and photochemical techniques. Substitution of chloride with bromide in the previously reported [{(phen)₂Ru(dpp)}₂RhCl₂](PF₆)₅ complex presents a new photoinitiated electron collector which can assist in understanding the functioning of our supramolecular systems [{(TL)₂Ru(BL)}₂RhX₂](PF₆)₅ (TL = terminal ligand, BL = bridging ligand, X = halide) in the photoinitiated electron collection and generation of hydrogen through the reduction of water and a detailed comparison is presented. Both the bromide and chloride analogues of these supramolecular complexes contain low energy, emissive metal-to-ligand charge transfer (³MLCT) excited states that populate lower lying metal-to-metal charge transfer (³MMCT) excited states. The electrochemistry of these complexes showed an impact on the reduction of the central Rh^III upon halide substitution with the bromide analogue [(phen)₂Ru(dpp)}₂RhBr₂](PF₆)₅ having a slightly lower reduction potential than the corresponding chloride counterpart. The more positive reduction of Rh^III to generate the Rh^I species in the bromide analogue impacts the photocatalytic properties upon photolysis in the presence of a sacrificial electron donor. The trimetallic complex [{(phen)₂Ru(dpp)}₂RhBr₂](PF₆)₅ generates hydrogen through the reduction of water with higher yields than the chloride [{(phen)₂Ru(dpp)}₂RhCl₂](PF₆)₅ analogue under the same conditions. Despite the longer lived ³MLCT state of both [(TL)₂Ru(dpp)]²⁺ and [{(TL)₂Ru}₂(dpp)]⁴⁺ when TL = phen vs. bpy (bpy = 2,2′-bipyridine), the phen trimetallics with X = Cl^? or Br^? do not display longer lived ³MLCT states and show lower H₂ yields than the analogous bpy trimetallic systems.     

Fluorescence and DNA-binding spectral studies of neodymium(III) complex containing 2,2′-bipyridine, [Nd(bpy)2Cl3·OH2]

The interaction of [Nd(bpy)₂Cl₃·OH₂], where bipy is 2,2′-bipyridine, with DNA has been studied by absorption, emission, and viscosity measurements. [Nd(bpy)₂Cl₃·OH₂] showed absorption decreasing in charge transfer band with increasing of DNA. The binding constant, K_b has been determined by absorption measurement and found to be (1.5 ± 0.1) × 10⁵ M^?1. The fluorescent of [Nd(bpy)₂Cl₃·OH₂] has been investigated in detail. The interaction was also studied by fluorescence quenching technique. The results of fluorescence titration revealed that DNA had the strong ability to quenching the intrinsic fluorescence of Nd(III) complex at 327 nm. The binding site number n, apparent binding constant K_b and the Stern–Volmer quenching constant K_SV have been determined. Thermodynamic parameters have been calculated according to relevant fluorescent data and Van’t Hoff equation. Characterization of bonding mode has been studied. The results suggested that the major interaction mode between [Nd(bpy)₂Cl₃·OH₂] and DNA was groove binding.     

Effect of intraligand π-delocalization on the photophysical properties of two new Ru(II) complexes

Two new Ru(II) complexes, [Ru(bpy)₂(1-COO-iqu)]⁺ (2; bpy = 2,2′-bipyridine, 1-COO-iqu^? = isoquinoline-1-carboxylate) and [Ru(bpy)₂(3-COO-iqu)]⁺ (3; 3-COO-iqu^? = isoquinoline-3-carboxylate), were prepared and their crystal structures solved. The ground and excited state properties of 2 and 3 were characterized and compared to those of [Ru(bpy)₃]²⁺ (1). The presence of the oxygen atom in the Ru(II) coordination sphere makes 2 and 3 easier to oxidize than 1. The Ru → bpy MLCT absorption and emission of 2 and 3 are red-shifted relative to that of 1 in CH₂Cl₂, and the E₀₀ energies were estimated to be 1.89 eV and 1.95 eV from the low temperature emission of 2 and 3, resulting in excited state oxidation potentials of ?1.03 V and ?1.10 V vs SCE, respectively. In addition to the short-lived emissive ³MLCT state, a long-lived species is observed in the transient absorption of 3 in DMSO (τ = 49 μs) and pyridine (τ = 44 μs), assigned to a solvent-coordinated complex. This intermediate is not observed for 3 in non-polar solvents or for 2. The absence of the solvent coordinated intermediate in 2 is explained by the stronger Ru–O bond afforded by the lower conjugation in that extends onto the carboxylic acid in the 1-COO-iquo^?ligand, compared to that in the 3-COO-iqu^?ligand in 3. Transient absorption experiments also show that the ³MLCT excited state of 3 is able to reduce methyl viologen.     

Coupled chemoenzymatic transfer hydrogenation catalysis for enantioselective reduction and oxidation reactions

Stereoselective reductions of prochiral ketones were performed using a new thermophilic, NAD-dependent alcohol dehydrogenase from Thermus sp. (TADH). The enzyme was produced on 2L-scale from recombinant Escherichia coli and purified by a simple, one-step heat treatment procedure yielding 220 mg of pure enzyme. Regeneration of NADH was catalyzed by the organometallic complex [Cp*Rh(bpy)(H₂O)]²⁺ using formate as a reducing agent. The catalytic performance of [Cp*Rh(bpy)(H₂O)]²⁺ in terms of total number of catalytic cycles and number of catalytic cycles per hour achieved herein (up to 1500 and more than 400 h⁻¹, respectively), are the highest reported for a non-enzymatic nicotinamide regeneration system so far. Chemoenzymatic reduction reactions in a two liquid phase setup were performed on a gramme-scale, for example, 1.3 g of enantiopure (1S,3S)-3-methylcyclohexanol was obtained after purification. The volumetric productivity reached up to 3.9 mM h⁻¹ with an average of 2.6 mM h⁻¹ (5.3 g L⁻¹ d⁻¹) over 10 h. In addition, chemoenzymatic oxidations utilizing the same catalyst set and molecular oxygen as a terminal electron acceptor were performed. Thus, the preparative value of chemoenzymatic transfer hydrogenations with [Cp*Rh(bpy)(H₂O)]²⁺ as a regeneration catalyst coupled especially to thermophilic ADHs was demonstrated.     

Bioelectrocatalytic oxidation of glucose by hexose oxidase directly wired to graphite

Glucose-oxidizing enzymes are widely used in electrochemical biosensors and biofuel cells; in most applications glucose oxidase, an enzyme with non-covalently bound FAD and low capability of direct electronic communications with electrodes, is used. Here, we show that another glucose-oxidizing enzyme with a covalently bound FAD center, hexose oxidase (HOX), adsorbed on graphite, exhibits a pronounced non-catalytic voltammetric response from its FAD, at − 307 mV vs. Ag/AgCl, pH 7, characterized by the heterogeneous electron transfer (ET) rate constant of 29.2 ± 4.5 s^− 1. Direct bioelectrocatalytic oxidation of glucose by HOX proceeded, although, with a 350 mV overpotential relative to FAD signals, which may be connected with a limiting step in biocatalysis under conditions of the replacement of the natural redox partner, O₂, by the electrode; mediated bioelectrocatalysis was consistent with the potentials of a soluble redox mediator used. The results allow development of HOX-based electrochemical biosensors for sugar monitoring and biofuel cells exploiting direct ET of HOX, and, not the least, fundamental studies of ET non-complicated by the loss of FAD from the protein matrix.     

Synthesis and characterization of three Cu(II), Zn(II), Cd(II) supramolecular complexes bridged by biphenyl 2,2′-dicarboxylate

Three complexes, M₂(bpy)₂(bpdc)₂·xH₂O [M = Cu, x = 0; M = Zn or Cd, x = 2], have been hydrothermally synthesized by 1,1′-biphenyl-2,2′-dicarboxylic acid (H₂bpdc) with 2,2′-bipyridine (bpy) to form binuclear molecules. In each, the two bpdc groups align the two opposing planar [M(bpy)]²⁺ cations. The molecules are connected by C–H⋯O hydrogen bonds, π–π stacking, and C–H⋯π interactions to form three dimensional supramolecular networks. Furthermore, at room temperature, complex 3 exhibits strong photoluminescence.     

Synthesis of (NHC)Rh(cod)Cl and (NHC)RhCl(CO)2 complexes – Translation of the Rh- into the Ir-scale for the electronic properties of NHC ligands

Twenty-three different Rh complexes of the (NHC)RhCl(cod) and (NHC)RhCl(CO)₂ type were synthesized from [RhCl(cod)]₂. The electron donating nature of the NHC ligands was changed in a systematic manner. The redox potentials of the various (NHC)RhCl(cod) and the ν(CO) of the various (NHC)RhCl(CO)₂ were determined. A correlation of the Rh redox potentials and the Rh ν(CO), respectively, with the related data from analogous (NHC)IrCl(cod) and (NHC)IrCl(CO)₂ complexes established two linear relationships. The linear regression (R² = 0.993) of the Rh and the Ir redox potentials results in an equation for the redox potential transformation: E_1/2(Ir) = 1.016 · E_1/2(Rh) ? 0.076 V. The linear regression (R² = 0.97) of the Rh and Ir ν_av(CO) results in an equation for the ν_av(CO) transformation: ν_av(CO)Ir = 0.8695 · ν_av(CO)Rh + 250.7 cm^?1. In this manner the Rh and the Ir-scale for the determination of the electron donating properties of NHC ligands are unified.     

Synthesis,characterizations of silica-coated gold nanorods and its applications in electroanalysis of hemoglobin

Gold nanorods (GNRs) were synthesized by a seed–mediated growth approach followed by TEOS polymerization leading to the formation of silica layer surrounding the gold nanorod core. TEM images showed that the silica-coated gold nanorods (GNRs@SiO₂) were dispersed with an average aspect ratio of 3.1 for the GNRs cores and a uniform thickness of the silica shell. The core/shell nanocomposites were further used as efficient supports for the immobilization of hemoglobin (Hb) to fabricate a novel biosensor. The immobilized Hb showed an enhanced electron transfer for its heme Fe(III) to Fe(II) redox couple. This biosensor showed an excellent bioelectrocatalytic activity towards H₂O₂ with a linear range from 8.0 × 10⁻⁷ to 6.1 × 10⁻⁵ M, and the detection limit was 6.0 × 10⁻⁸ M at 3σ. The apparent Michaelis–Menten constant of the immobilized hemoglobin was calculated to be 0.13 mM.     

Complexes derived from the copper(II) perchlorate/maleamic acid/2,2′-bipyridine and copper(II) perchlorate/maleic acid/2,2′-bipyridine reaction systems: Synthetic,reactivity, structural and spectroscopic studies

A systematic investigation of the reactions of Cu(ClO₄)₂ · 6H₂O with maleamic acid (H₂L) in the presence of 2,2′-bipyridine (bpy) has been carried out. The chemical and structural identity of the products depends on the solvent, the absence or presence of external hydroxides in the reaction mixture and the molar ratio of the reactants. Various reaction schemes have led to the isolation of the complexes [Cu₂(HL)₂(bpy)₂(H₂O)₂](ClO₄)₂ (1), [Cu₂(HL)₂(bpy)₂(H₂O)₂](ClO₄)₂ · 2H₂O (1 · 2H₂O), [Cu(L′′)(bpy)]_n · 2nH₂O (2 · 2nH₂O), [Cu₂(L′′)(bpy)₂(H₂O)₂]_n(ClO₄)_2n · 0.5nH₂O (3 · 0.5nH₂O), [Cu₂(L′′)₂(bpy)₂] · 2MeOH (5 · 2MeOH), [Cu₂(L′)₂(bpy)₂(ClO₄)₂] (6) and [Cu(ClO₄)₂(bpy)(MeCN)₂] (7b), where L′′^2? and L′^? are the maleate(?2) and monomethyl maleate(?1) ligands, respectively. The HL^? ion has been transformed to L′′^2? and L′^? in the known compounds 2 · 2nH₂O and 6, respectively, via metal ion-assisted processes involving hydrolysis (2 · 2nH₂O) and methanolysis (6) of the primary amide group. The reaction that leads to 6 takes place through the formation of the mononuclear complex [Cu(ClO₄)₂(bpy)(MeOH)₂] (7a), whose structure was assigned on the basis of its spectral similarity with the structurally characterized complex 7b. The structures of the cations in 1 and 1 · 2H₂O consists of two Cu^II atoms bridged by the carboxylate groups of the two HL^? ligands, each exhibiting the less common η² coordination mode; a chelating bpy molecule and a H₂O ligand complete square pyramidal coordination at each metal centre. The structure of the dinuclear repeating unit in the 1D coordination polymer 3 · 0.5nH₂O consists of two Cu^II atoms bridged by two syn,syn η¹:η¹:μ₂ carboxylate groups belonging to two L′′^2? ions; each ligand bridged two neighboring [Cu^II,II₂] units thus promoting the formation of a helical chain. The structure of the dinuclear molecule of complex 5 · 2MeOH consists of two Cu^II atoms bridged by two η² carboxylate groups from two L′′^2? ligands; the second carboxylate group of each maleate(?2) ligand is monodentately coordinated to Cu^II, creating a remarkable seven-membered chelating ring. The L′^? ion behaves as a carboxylate-type ligand in 6, with the carboxylate group being in the familiar syn,syn η¹:η¹:μ₂ coordination mode; a chelating bpy molecule and a coordinated ClO₄^? complete five-coordination at each Cu^II centre. The crystal structures of the complexes are stabilized by various H-bonding patterns. Characteristic IR bands of the complexes are discussed in terms of the known structures and the coordination modes of the ligands.     

Enhancement of electrochemical and catalytic properties of MoS2 through ball-milling

MoS₂ nanosheets of one to few layer thickness present novel electronic and enhanced catalytic properties with respect to the bulk material. Here we show that a simple and highly scalable ball-milling procedure can lead to significant improvements of the electrochemical and catalytic properties of the bulk natural MoS₂. We characterized the material before and after the milling process by means of scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy in order to evaluate morphological and chemical features. We investigated the electrochemical properties by means of voltammetry techniques to monitor the electron transfer with [Fe(CN)₆]^{4 −/3 −} redox probe and the catalytic properties by monitoring the electrochemical hydrogen evolution reaction (HER). A significant overpotential lowering of about 210 mV is obtained for the HER by the ball-milled material when compared to bulk materials. This has a huge potential for the lowering of the energy consumption during hydrogen evolution. Ball-milling offers highly scalable dry method for large scale production of electrocatalyst with enhanced properties.     

Direct electrochemistry and electrocatalysis of hemoglobin entrapped in composite matrix based on chitosan and CaCO3 nanoparticles

The direct electron transfer between hemoglobin (Hb) and the underlying glassy carbon electrode (GCE) can be readily achieved via a high biocompatible composite system based on biopolymer chitosan (CHT) and inorganic CaCO₃ nanoparticles (nano-CaCO₃). Cyclic voltammetry of Hb-CHT/nano-CaCO₃/GCE showed a pair of stable and quasi-reversible peaks for HbFe(III)/Fe(II) redox couple in pH 7.0 buffer. The electrochemical reaction of Hb immobilized in CHT/nano-CaCO₃ composite matrix exhibited a surface-controlled process accompanied by electron and proton transfer. The electron transfer rate constant was estimated to be 1.8 s⁻¹. This modified electrode showed a high thermal stability up to 60 °C. The apparent Michaelis–Menten constant was calculated to be 7.5 × 10⁻⁴ M, indicating a high catalytic activity of the immobilized Hb toward H₂O₂. The interaction between Hb and this nano-hybrid material was also investigated using FT-IR and UV–vis spectroscopy, indicating that Hb retained its native structure in this hybrid matrix.     

Pulse radiolysis study of the formation and the reactivity of baicalin radical anion,and in comparison with rutin,quercetin and acyrlate ester radical anions in ethanol

The reaction of solvated electrons with baicalin in N₂-saturated ethanol has been studied by pulse radiolysis. The results show that a solvated electron can add to baicalin and generate a baicalin radical anion with a maximum UV absorbance peak at 360 nm. Its molar extinction coefficient at this wavelength is 1.3×10⁴ M⁻¹ cm⁻¹. The rate constant for the build-up of the baicalin radical anion is 1.3(±0.4)×10¹⁰ M⁻¹ s⁻¹. Decay of the radical anion is induced by a proton transfer reaction and a recombination reaction, which involves a pseudo-first-order reaction with rate constant 2.6(±0.4)×10³ s⁻¹ and a second-order reaction with rate constant 1.3(±0.2)×10⁹ M⁻¹ s⁻¹. The effect of acetaldehyde on the decay of the baicalin radical anion was also investigated. Electron transfer between the baicalin radical anion and acetaldehyde was not observed, probably due to the low rate of electron transfer between the baicalin radical anion and acetaldehyde. Reactivity of the rutin, quercetin, baicalin and ethyl acrylate radical anions are also compared.     

ATP- and redox-induced conformational changes in the activator of the radical enzyme 2-hydroxyisocaproyl-CoA dehydratase

A [4Fe–4S]¹⁺ cluster-containing protein activates 2-hydroxyisocaproyl-CoA dehydratase by an ATP-driven electron transfer. The activator has been proposed to change its conformation by MgATP similarly to nitrogenase Fe-protein. Iron chelation by bathophenanthroline removed the reduced [4Fe–4S]¹⁺ cluster from the activator in an ATP-dependent manner (rate, v = 0.128 ± 0.004 min⁻¹; K_m = 21 ± 1 μM); with ADP no chelation was observed (v < 0.001 min⁻¹). Chelation of the oxidised [4Fe–4S]²⁺ cluster occurred faster with ADP (v = 0.34 ± 0.05 min⁻¹) than with ATP (v = 0.132 ± 0.005 min⁻¹). The data indicate that reduction of the activator and binding of ATP induce conformational changes necessary to transfer the electron to the dehydratase. Interaction of both proteins promotes ATP hydrolysis (K_m = 0.5 ± 0.1 μM).     

Controlling the dimensionality of oxalate-based bimetallic complexes: The ferromagnetic chain {[K(18-crown-6)][Mn(bpy)Cr(ox)3]}∞(18-crown-6 = C12H24O6, ox=C2O42-, bpy = C10H8N2)

The bimetallic ferromagnetic chain {[K(18-crown-6)][Mn(bpy)Cr(ox)₃]}_∞ (1) has been synthesized and characterized. It crystallizes in the orthorhombic chiral space group P2₁2₁2₁ [a = 9.0510(2) Å, b = 14.4710(3) Å, c = 26.8660(8) Å, V = 3510.97(1) Å³, Z = 2]. Compound 1 is made up by anionic [Mn(bpy)Cr(ox)₃]⁻ 1D chains and cationic [K(18-crown-6)]⁺ complexes. The magnetic exchange within the chain is ferromagnetic [J = +7.8(7) cm⁻¹]. In the solid state, the ferromagnetic chains are well isolated magnetically and no long range magnetic ordering has been observed above 2 K.     

A molecular wire modified glassy carbon electrode for achieving direct electron transfer to native glucose oxidase

Here we describe a strategy for achieving direct electron transfer to native glucose oxidase (GOx), an enzyme in which the redox active centre is buried deep within the glycoprotein. To achieve this a glassy carbon electrode is modified with a mixed monolayer of 4-carboxyphenyl and a 20 Å long oligo(phenylethynyl) molecular wire (MW), assembled from the respective aryl diazonium salts. Subsequently GOx is adsorbed to the interface, followed by covalent attachment. The redox chemistry of the active centre of glucose oxidase, flavin adenine dinucleotide, was observed at an E_1/2 of –443 mV (vs. Ag|AgCl). The enzyme was shown to retain its activity. Most importantly, in the absence of oxygen the electrode was still able to biocatalytically turn over glucose at −400 mV, thereby demonstrating that the enzyme was being recycled back to its catalytically active oxidized form from its inactive reduced form. The rate of enzyme turnover was 1.1 s⁻¹.     

Direct electrochemistry and electrocatalysis of hemoglobin in sodium alginate film on a BMIMPF6 modified carbon paste electrode

A room temperature ionic liquid (RTIL) modified carbon paste electrode was constructed based on the substitute of paraffin with 1-butyl-3-methyl-imidazolium hexafluorophosphate (BMIMPF₆) as binder for carbon paste. Direct electrochemistry and electrocatalytic behaviors of hemoglobin (Hb) entrapped in the sodium alginate (SA) hydrogel film on the surface of this carbon ionic liquid electrode (CILE) were investigated. The presence of IL in the CILE increased the electron transfer rate and provided a biocompatible interface. Hb remained its bioactivity on the surface of CILE and the SA/Hb modified electrode showed a pair of well-defined, quasi-reversible cyclic voltammetric peaks with the apparent standard potential (E^0′) at about −0.344 V (vs. SCE) in pH 7.0 Britton–Robinson (B–R) buffer solution, which was attributed to the Hb Fe(III)/Fe(II) redox couple. UV–Vis absorption spectra indicated that heme microenvironment of Hb in SA film was similar to its native status. Hb showed a thin-layer electrochemical behavior in the SA film with the direct electron transfer achieved on CILE without the help of electron mediator. Electrochemical investigation indicated that Hb took place one proton with one electron electrode process and the average surface coverage of Hb in the SA film was 3.2 × 10⁻¹⁰ mol/cm². The immobilized Hb showed excellent electrocatalytic responses to the reduction of H₂O₂ and nitrite.