Enzyme Immobilization and Direct Electrochemistry Based on a New Matrix of Phospholipid‐Monolayer‐Functionalized Graphene

A new nanocomposite material for enzyme immobilization and subsequent direct electrochemistry and electrocatalysis was developed by using 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1‐rac‐glycerol)‐phospholipid‐monolayer‐membrane‐modified graphene (DMPG‐G). Microperoxidase‐11 (MP11) was chosen as a model enzyme to investigate the composite system. Owing to the improved conductivity and biocompatible microenvironment, MP11 that was immobilized in the matrix of the DMPG‐G nanocomposite (DMPG‐G‐MP11) effectively retained its native structure and bioactivity. DMPG‐G‐MP11‐modified glassy carbon electrode (DMPG‐G‐MP11/GCE) exhibited a pair of well‐defined quasi‐reversible redox peaks of MP11 and showed high electrocatalytic activity towards hydrogen peroxide (H₂O₂). The linear response of the developed biosensor for the determination of H₂O₂ ranged from 2.0×10^?6 to 4.5×10^?4 M with a detection limit of 7.2×10^?7 M . This biosensor exhibited high reproducibility and long‐term storage stability. The promising features of this biosensor indicate that these lipid–graphene nanocomposites are ideal candidate materials for the direct electrochemistry of redox proteins and that they could serve as a versatile platform for the construction of a third‐generation biosensor.     

A Hydrogen Peroxide Biosensor Based on Room Temperature Ionic Liquid Functionalized Graphene Modified Carbon Ceramic Electrode

A room temperature ionic liquid (IL) 1‐butyl‐3‐methylimidazolium hexafluorophosphate functionalized graphene (GE) was prepared and a hydrogen peroxide (H₂O₂) biosensor was fabricated by immobilizing hemoglobin (Hb) into the IL‐GE composite film. UV‐visible and Fourier transform infrared spectra of the composite film indicated that Hb retained its native structure in the film. Electrochemical investigation of the biosensor showed a pair of well‐defined, quasi‐reversible redox peaks with E_pa=?0.209 V and E_pc= ?0.302 V (vs. SCE) in pH 7.0 phosphate buffer solution at the scan rate of 100 mV/s. To the reduction of H₂O₂, the biosensor had a good linear range from 8.0×10^?7 to 1.8×10^?4 mol/L with a detection limit of 3.0×10^?7 mol/L. The apparent Michaelis‐Menten constant K^app_M was estimated to be 3.4×10^?5 mol/L.     

Direct Electrochemistry of Hemoglobin Entrapped in Composite Electrodeposited Chitosan‐Multiwall Carbon Nanotubes and Nanogold Particles Membrane and Its Electrocatalytic Application

Hemoglobin was entrapped in composite electrodeposited chitosan‐multiwall carbon nanotubes (MCNTs) film by assembling gold nanoparticles and hemoglobin step by step. In phosphate buffer solution (pH 7), a pair of well‐defined and quasireversible redox peaks appeared with formal potential at ?0.289 V and peak separation of 100 mV. The redox peaks respected for the direct electrochemistry of hemoglobin at the surface of chitosan‐MCNTs‐gold nanoparticles modified electrode. The parameters of experiments have also been optimized. The composite electrode showed excellent electrocatalysis to peroxide hydrogen and oxygen, the peak current was linearly proportional to H₂O₂ concentration in the range from 1×10^?6 mol/L to 4.7×10^?4 mol/L with a detection limit of 5.0×10^?7 mol/L, and this biosensor exhibited high stability, good reproducibility and better selectivity. The biosensor showed a Michaelis–Menten kinetic response as H₂O₂ concentration is larger than 5.0×10^?4 mol/L, the apparent Michaelis–Menten constant for hydrogen peroxide was calculated to be 1.61 μmol/L.     

Cobalt(II) Schiff Base/Large Mesoporous Carbon Composite Film Modified Electrode as Electrochemical Biosensor for Hydrogen Peroxide and Glucose

A simple method was developed to prepare a cobalt(II) Schiff base (Co(salen))/large mesoporous carbon (LMC) composite film. The structure and electrocatalytic performance of the Co(salen)/LMC film were investigated by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The Co(salen)/LMC film exhibits high electrocatalytic activity toward H₂O₂, such as low detection limit (8.5×10^?7 M) and wide linear concentration range (2.0×10^?6–8.9×10^?3 M). Furthermore, glucose oxidase (GOD) was self‐assembled on the surface of the Co(salen)/LMC film modified electrode. Determination of glucose in human blood serum with satisfying result was investigated by the resulting biosensor.     

Amperometric Glucose Biosensor Based on Glucose Oxidase Encapsulated in Carbon Nanotube–Titania–Nafion Composite Film on Platinized Glassy Carbon Electrode

A highly sensitive and selective glucose biosensor has been developed based on immobilization of glucose oxidase within mesoporous carbon nanotube–titania–Nafion composite film coated on a platinized glassy carbon electrode. Synergistic electrocatalytic activity of carbon nanotubes and electrodeposited platinum nanoparticles on electrode surface resulted in an efficient reduction of hydrogen peroxide, allowing the sensitive and selective quantitation of glucose by the direct reduction of enzymatically‐liberated hydrogen peroxide at ?0.1 V versus Ag/AgCl (3 M NaCl) without a mediator. The present biosensor responded linearly to glucose in the wide concentration range from 5.0×10^?5 to 5.0×10^?3 M with a good sensitivity of 154 mA M^?1cm^?2. Due to the mesoporous nature of CNT–titania–Nafion composite film, the present biosensor exhibited very fast response time within 2 s. In addition, the present biosensor did not show any interference from large excess of ascorbic acid and uric acid.     

Hydrogen Peroxide Biosensor Based on Immobilization of Hemoglobin on Au@Ag Nanoparticles Modified Carbon Ionic Liquid Electrode

A hydrogen peroxide (H₂O₂) biosensor based on the combination of Au@Ag core‐shell nanoparticles with a hemoglobin‐chitosan‐1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (Hb‐CHIT‐BMIM×BF₄) composite film was prepared. UV‐vis spectroscopy and transmission electron microscopy confirmed a core‐shell nanostructure of Au@Ag nanoparticle was successfully obtained. Cyclic voltammetric results showed a pair of well‐defined redox peaks appeared with the formal potential (E^O′) of ‐0.301 V (versus Ag/AgCl reference electrode) and the peak‐to‐peak separation (ΔE_p) was 84 mV in 0.1 M phosphate buffer solutions. Due to the synergetic effect of Au@Ag core‐shell nanoparticles and Hb‐CHIT‐BMIM×BF₄, the biosensor exhibited good electrocatalytic activity to the reduction of H₂O₂ in a linear range from 1.0 × 10^?6 to 1.0 × 10^?3 M with a detection limit of 4 × 10^?7 M (S/N = 3). The apparent Michaelis‐Menten constant (K_M) was estimated to be 4.4 × 10^?4 M, showing its high affinity. Thus, the study proved that the combination of Au@Ag core‐shell nanoparticles and Hb‐CHIT‐BMIM×BF₄ is able to open up new opportunities for the design of enzymatic biosensors.     

Reduced Graphene Oxide|Carbon Ceramic Electrode Modified with CdS‐Hemoglobin as a Sensitive Hydrogen Peroxide Biosensor

A sensitive hydrogen peroxide (H₂O₂) biosensor was developed based on a reduced graphene oxide|carbon ceramic electrode (RGO|CCE) modified with cadmium sulfide‐hemoglobin (CdS‐Hb). The electron transfer kinetics of Hb were promoted due to the synergetic function of RGO and CdS nanoparticles. The transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) were calculated to be 0.54 and 2.6 s^?1, respectively, indicating a great facilitation achieved in the electron transfer between Hb and the electrode surface. The biosensor showed a good linear response to the reduction of H₂O₂ over the concentration range of 2–240 µM with a detection limit of 0.24 µM (S/N=3) and a sensitivity of 1.056 µA µM^?1 cm^?2. The high surface coverage of the CdS‐Hb modified RGO|CCE (1.04×10^?8 mol cm^?2) and a smaller value of the apparent Michaelis? Menten constant (0.24 mM) confirmed excellent loading of Hb and high affinity of the biosensor for hydrogen peroxide.     

Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on Fe3O4/Chitosan Modified Glassy Carbon Electrode

A new type of amperometric hydrogen peroxide biosensor was constructed based on horseradish peroxidase (HRP) immobilized on Fe₃O₄/chitosan modified glassy carbon electrode. The effects of some experimental variables such as the concentration of supporting electrolyte, pH, enzyme loading, the concentration of the mediator of methylene blue (MB) and the applied potential were investigated. The linear range of the calibration curve for H₂O₂ was 2.0×10^?4–1.2×10^?2 M with a detection limit of 1.0×10^?4 M (S/N=3). The response time was less than 12 s. The apparent Michaelis‐Menten constant K_m was 21.4 mM and it illustrated the excellent biological activity of the fixed enzyme. In addition, the biosensor had long‐time stability and good reproducibility. And this method has been used to determine H₂O₂ concentration in the real sample.     

Immobilization and Bioelectrochemistry of Hemoglobin Based on Carrageenan and Room Temperature Ionic Liquid Composite Film

A novel biopolymer/room‐temperature ionic liquid composite film based on carrageenan, room temperature ionic liquid (IL) [1‐butyl‐3‐methylimidazolium tetra?uoroborate ([BMIM]BF₄)] was explored for immobilization of hemoglobin (Hb) and construction of biosensor. Direct electrochemistry and electrocatalytic behaviors of Hb entrapped in the IL‐carrageenan composite ?lm on the surface of glassy carbon electrode (GCE) were investigated. UV‐vis spectroscopy demonstrated that Hb in the IL‐carrageenan composite ?lm could retain its native secondary structure. A pair of well‐de?ned redox peaks of Hb was obtained at the Hb‐IL‐carrageenan composite ?lm modi?ed electrode through direct electron transfer between the protein and the underlying electrode. The heterogeneous electron transfer rate constant (k_s) was 2.02 s^?1, indicating great facilitation of the electron transfer between Hb and IL‐carrageenan composite film modi?ed electrode. The modi?ed electrode showed excellent electrocatalytic activity toward reduction of hydrogen peroxide with a linear range of 5.0×10^?6 to 1.5×10^?4 mol/L and the detection limit was 2.12×10^?7 mol/L (S/N=3). The apparent Michaelis‐Menten constant K_M^app for hydrogen peroxide was estimated to be 0.02 mmol/L, indicating that the biosensor possessed high af?nity to hydrogen peroxide. In addition, the proposed biosensor showed good reproducibility and stability.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Electrogenerated Chemiluminescence Ethanol Biosensor Based on Carbon Nanotube‐Titania‐Nafion Composite Film

Mesoporous titania‐Nafion composite doped with carbon nanotube (CNT) has been used for the immobilization of tris(2,2′‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) and alcohol dehydrogenase on an electrode surface to yield a highly sensitive and stable electrogenerated chemiluminescence (ECL) ethanol biosensor. The presence of CNT in the composite film increases not only the sensitivity of the ECL biosensor but also the long‐term stability of the biosensor. The present biosensor responds linearly to ethanol in the wide concentration ranges from 1.0×10^?5 M to 1.0×10^?1 M with a detection limit of 5.0×10^?6 M (S/N=3). The present ECL ethanol biosensor exhibited higher ECL response compared to that obtained with the ECL biosensor based on the corresponding composite without CNT. The present CNT‐based ECL biosensor showed good long‐term stability with 75% of its initial activity retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

Sensitive and Selective Sensing of Hydrogen Peroxide with Iron‐Tetrasulfophthalocyanine‐Graphene‐Nafion Modified Screen‐Printed Electrode

We developed a novel iron‐tetrasulfophthalocyanine‐graphene‐Nafion (FeTSPc‐GR‐Nafion) modified screen‐printed electrode to determine hydrogen peroxide (H₂O₂) with high sensitivity and selectivity. The nanocomposite film (FeTSPc‐GR‐Nafion) exhibits an excellent electrocatalytic activity towards oxidation of H₂O₂ at a potential of +0.35 V in the absence of enzyme. A comparative study reveals that the FeTSPc‐GR complexes play a dual amplification role. Amperometric experiment indicates that the sensors possess good sensitivity and selectivity, with a linear range from 2.0×10^?7 M to 5.0×10^?3 M and a detection limit of 8.0×10^?8 M. This sensor has been successfully used to develop the glucose biosensor and has also been applied to determine H₂O₂ in sterile water.     

An Organic Sol‐Gel Film as Modifier to Construct Biosensor

A new amperometric biosensor for hydrogen peroxide (H₂O₂) was developed by adsorbing hemoglobin (Hb) on an organic sol‐gel film. The organic sol‐gel was prepared using resorcinol and formaldehyde as monomers. This sol‐gel film shows a biocompatible microenvironment for retaining the native activity of the adsorbed Hb. The direct electron transfer between Hb and electrode is achieved. Hb adsorbed on the film shows an enzyme‐like catalytic activity for the reduction of H₂O₂. The reduction peak currents are proportional linearly to the concentration of hydrogen peroxide in the range of 6×10^?8 to 3.6×10^?6 M, with a detection limit of 2.4×10^?8 M (S/N=3). This research enlarges the applications of organic sol‐gel materials in biosensor field.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin Based on Nafion‐Room Temperature Ionic Liquids‐Multiwalled Carbon Nanotubes Composite Film

A robust and effective composite film combined the benefits of Nafion, room temperature ionic liquid (RTIL) and multi‐wall carbon nanotubes (MWNTs) was prepared. Hemoglobin (Hb) was successfully immobilized on glassy carbon electrode surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Hb were investigated in detail. A pair of well‐defined and quasi‐reversible redox peaks of Hb was obtained in 0.10 mol·L^?1 pH 7.0 phosphate buffer solution (PBS), indicating that the Nafion‐RTIL‐MWNTs film showed an obvious promotion for the direct electron transfer between Hb and the underlying electrode. The immobilized Hb exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis current was linear to H₂O₂ concentration in the range of 2.0×10^?6 to 2.5×10^?4 mol·L^?1, with a detection limit of 8.0×10^?7 mol·L^?1 (S/N=3). The apparent Michaelis‐Menten constant (K_m^app) was calculated to be 0.34 mmol·L^?1. Moreover, the modified electrode displayed a good stability and reproducibility. Based on the composite film, a third‐generation reagentless biosensor could be constructed for the determination of H₂O₂.     

Multilayer Assembly of Hemoglobin and Colloidal Gold Nanoparticles on Multiwall Carbon Nanotubes/Chitosan Composite for Detecting Hydrogen Peroxide

Chitosan (CS) was chosen for dispersing multi‐wall carbon nanotubes (MWNTs) to form a stable CS‐MWNTs composite, which was first coated on the surface of a glassy carbon electrode to provide a containing amino groups interface for assembling colloidal gold nanoparticles (GNPs), followed by the adsorption of hemoglobin (Hb). Repeating the assembly step of GNPs and Hb resulted in {Hb/GNPs}_n multilayers. The assembly of GNPs onto CS‐MWNTs composites was confirmed by transmission electron microscopy. The consecutive growth of {Hb/GNPs}_n multilayers was confirmed by cyclic voltammetry and UV‐vis absorption spectroscopy. The resulting system brings a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of GNPs and MWNTs. The resulting biosensor displays an excellent electrocatalytic activity and rapid response for hydrogen peroxide. The linear range for the determination of H₂O₂ was from 5.0×10^?7 to 2.0×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelis–Menten constant K_M^app value of 0.19 mM.     

Noncovalent Assembly of Picket‐Fence Porphyrins on Nitrogen‐Doped Carbon Nanotubes for Highly Efficient Catalysis and Biosensing

A water‐insoluble picket‐fence porphyrin was first assembled on nitrogen‐doped multiwalled carbon nanotubes (CN_x MWNTs) through Fe? N coordination for highly efficient catalysis and biosensing. Scanning electron micrographs, Raman spectra, X‐ray photoelectron spectra, UV/Vis absorption spectra, and electrochemical impedance spectra were employed to characterize this novel nanocomposite. By using electrochemical methods on the porphyrin at low potential in neutral aqueous solution, the presence of CN_x MWNTs led to the direct formation of a high‐valent iron(IV)–porphyrin unit, which produced excellent catalytic activity toward the oxidation of sulfite ions. By using sulfite ions, a widely used versatile additive and preservative in the food and beverage industries, as a model, a highly sensitive amperometric biosensor was proposed. The biosensor showed a linear range of four orders of magnitude from 8.0×10^?7 to 4.9×10^?3 mol L^?1 and a detection limit of 3.5×10^?7 mol L^?1 due to the highly efficient catalysis of the nanocomposite. The designed platform and method had good analytical performance and could be successfully applied in the determination of sulfite ions in beverages. The direct noncovalent assembly of porphyrin on CN_x MWNTs provided a facile way to design novel biofunctional materials for biosensing and photovoltaic devices.     

Fabrication of Glucose Biosensor Based on Encapsulation of Glucose‐Oxidase on Sol‐Gel Composite at the Surface of Glassy Carbon Electrode Modified with Carbon Nanotubes and Celestine Blue

A simple procedure was developed to prepare a glassy carbon electrode modified with multi walled carbon nanotubes (MWCNTs) and Celestin blue. Cyclic voltammograms of the modified electrode show stable and a well defined redox couple with surface confined characteristic at wide pH range (2–12). The formal potential of redox couple (E′) shifts linearly toward the negative direction with increasing solution pH. The surface coverage of Celestine blue immobilized on CNTs glassy carbon electrode was approximately 1.95×10^?10 mol cm^?2. The charge transfer coefficient (α) and heterogeneous electron transfer rate constants (k_s) for GC/MWCNTs/Celestine blue were 0.43 and 1.26 s^?1, respectively. The modified electrode show strong catalytic effect for reduction of hydrogen peroxide and oxygen at reduced overpotential. The glucose biosensor was fabricated by covering a thin film of sol‐gel composite containing glucose oxides (GOx) on the surface of Celestine blue /MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 0.3 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. The accuracy of the biosensor for glucose detection was evaluated by detection of glucose in a serum sample, using standard addition protocol. In addition biosensor can reach 90% of steady currents in about 3.0 sec and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) was eliminated. Furthermore, the apparent Michaelis–Menten constant 2.4 mM, of GOx on the nano composite exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility of redox couple, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this procedure for modification of glucose biosensor.     

Electrochemical Biosensing Platforms Using Phthalocyanine‐Functionalized Carbon Nanotube Electrode

Iron‐phthalocyanines (FePc) are functionalized at multi‐walled carbon nanotubes (MWNTs) to remarkably improve the sensitivity toward hydrogen peroxide. We constructed a highly sensitive and selective glucose sensor on FePc‐MWNTs electrode based on the immobilization of glucose oxidase (GOD) on poly‐o‐aminophenol (POAP)‐electropolymerized electrode surface. SEM images indicate that GOD enzymes trapped in POAP film tend to deposit primarily on the curved tips and evenly disperse along the sidewalls. The resulting GOD^@POAP/FePc‐MWNTs biosensor exhibits excellent performance for glucose with a rapid response (less than 8 s), a wide linear range (up to 4.0×10^?3 M), low detection limits (2.0×10^?7 M with a signal‐to‐noise of 3), a highly reproducible response (RSD of 2.6%), and long‐term stability (120 days). Such characteristics may be attributed to the catalytic activity of FePc and carbon nanotube, permselectivity of POAP film, as well as the large surface area of carbon nanotube materials.     

Amperometric Biosensor for Hydrogen Peroxide Based on Electrodeposited Sub-micrometer Gold Modified Glassy Carbon Electrode

IntroductionAmperometricbiosensorofhydrogenperoxideisofpracticalimportancebecauseofitswideapplicationsinchemical,biological,clinical,environmentalandmanyotherfields.Forimprovementofsensor抯quality,vari-ouskindsofchemicalmodificationmethodshavebeendevelopedforreducingredoxoverpotentialsofH2O2atelectrodesurfaces,increasingthedetectionsensitivity,linearrange,stabilityandlivetime.Ithasbeenshownthattheuseofsub-micrometersizedmetalparticlessuchasPt-blackcansignificantlyimprovethequalityofthebiosens…     

Electrochemical Behavior of 8‐Azaguanine at DNA Langmuir–Blodgett Modified Glassy Carbon Electrode and Its Analytical Application

A highly sensitive electrochemical biosensor for the detection of trace amounts of 8‐azaguanine has been designed. Double stranded (ds)DNA molecules are immobilized onto a glassy carbon electrode surface with Langmuir–Blodgett technique. The adsorptive voltammetric behaviors of 8‐azaguanine at DNA‐modified electrode were explored by means of cyclic voltammetry and square wave voltammetry. Compared with bare glassy carbon electrode (GCE), the Langmuir–Blodgett film modified electrode can greatly improve the measuring sensitivity of 8‐azaguanine. Under the optimum experimental conditions, the Langmuir–Blodgett film modified electrode in pH 3.0 Britton–Robinson buffer solutions shows a linear voltammetric response in the range of 5.0×10^?8 to 1.0×10^?5 mol L^?1 with detection limit 9.0×10^?9 mol L^?1. The method proposed was applied successfully for the determination of 8‐azaguanine in diluted human urine with wonderful satisfactory.