A New Nanogold-Labeled Immunoresonance Scattering Spectral Probe for Determination of Trace IgG

A kind of 9 nm gold nanoparticles was prepared with the trisodium citrate and used to label goat anti-human IgG to obtain an IgG immunoresonance scattering spectral probe. In pH 5.8 buffer solution and in the presence of polyethylene glycol （PEG）, the immune reaction between gold-labeled goat anti-human IgG and IgG took place, and the resonance scattering intensity at 580 nm （I580nm） was enhanced greatly. The enhanced intensity AIRS is pro- portional to the IgG concentration from 1.3 to 1.5 X 10^3 ng.mL^-1, with a detection limit of 0.78 ng.mL ^-1. This assay showed high sensitivity and good selectivity for quantitative determination of IgG in human serum, with satisfactory results.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

免疫纳米金催化共振散射光谱法测定痕量IgM

用粒径为10 nm的金纳米微粒标记羊抗人免疫球蛋白M（IgM）,制备了IgM的免疫纳米金共振散射光谱探针。在pH4.49的KH2PO4-Na2HPO4缓冲溶液及PEG存在下,金标羊抗人IgM与IgM发生特异性结合生成胶体金免疫复合物,离心分离,获得未反应的金标抗上层清液。以此纳米金标抗作为催化剂,在pH 1.93的盐酸-柠檬酸钠缓冲溶液,催化NH2OH·HCl还原吸附在免疫纳米金表面的金络离子物种（AuCl4-）生成粒径更大的金纳米微粒,导致580 nm 处金纳米微粒的共振散射强度急剧增大。结果表明,随着IgM浓度增大,离心上层液中金标抗降低,I 580 nm线性降低,其△I580 nm与IgM浓度在0.06～4.80 ng· ml-1范围内呈良好的线性关系,其回归方程为ΔI580 nm=14.5cIgM + 1.8,检出限为0.03 ng·ml-1。本法具有灵敏、快速和较高的特异性,用于定量分析人血清中IgM,结果满意。     

Electroanalytical Determination of Carcinoembryonic Antigen at a Silica Nanoparticles/Titania Sol–Gel Composite Membrane‐Modified Gold Electrode

A highly hydrophilic and nontoxic colloidal silica nanoparticle/titania sol–gel composite membrane was prepared on a gold electrode via a chemical vapor deposition method. With carcinoembryonic antigen (CEA) as a model antigen and encapsulation of carcinoembryonic antibody (anti‐CEA) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. The presence of silica nanoparticles provided a congenial microenvironment for adsorbed biomolecules. The formation of immunoconjugate by a simple one‐step immunoreaction between CEA in sample solution and the immobilized anti‐CEA introduced the change in the potential. The modified procedure was further characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Compared to the commonly applied methods, i.e., the TiO₂ direct embedding procedure, this strategy could allow for antibodies immobilized with higher loading amount and better retained immunoactivity. The resulting immunosensor exhibited high sensitivity, good precision, acceptable stability, accuracy, reproducibility and wide linear range from 1.5 to 240 ng mL^?1 with a detection limit of 0.5 ng mL^?1 at 3σ. 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 CEA in the clinical diagnosis. Furthermore, this composite membrane could be used efficiently for the entrapment of other biomarkers and clinical applications.     

Application of Colloidal Gold to Fluorophotometric Determination of Human IgG

A method for the determination of human immunoglobulin G(IgG) based on a colloidal gold label by fluorospectrophotometry was developed. The sandwich immunoreaction among goat-anti-human IgG, human IgG and goat-anti-human IgG labeled with colloidal gold nanoparticles was applied in this experiment. First, a sandwich immunocomplex was formed on the surface of 96 well clear polystyrene high bind stripwellTM microplate. After the formation of the sandwich immunocomplex, a solution was added to dissociate the immunocomplex at room temperature. Then the solution of each well was transferred into the corresponding test tube. Thirdly, the rhodamine B solution was injected into each test tube. The rhodamine B chloraurate was extracted into ether that was measured with a spectrofluorometer. The experimental results indicate that the fluorescence intensity increased with the increase of human IgG concentration. The fluorescence intensity of rhodamine B chloraurate at 570 nm was proportional to the logarithm of human IgG concentration in a range from 10 to 5×105 ng/mL. It was shown that the determination of human IgG was easily made with the proposed fluorospectrophotometry.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

A Novel Chemiluminescence Immunoassay Using Solid‐Phase Antigen for Free 17β‐Estradiol in Human Serum

A high‐performance chemiluminescence immunoassay, with long‐term durability, good precision and time‐saving, was proposed for the detection of free 17β‐estradiol (E₂) in human serum. Ninety‐six microplates were coated with bovine serum albumin conjugated E₂ antigen as solid phase for the immunoassay. The E₂‐BSA antigen coated on the microplate and the E₂ antigen in the sample competed for the binding sites on the horseradish peroxidase (HRP) labeled anti‐E₂ antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol‐H₂O₂ substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94×10^?3 µg·L^?1 in a linear detection range from 0.01 to 1.00 µg·L^?1, good recoveries between 105% and 108%, and high precision with intra‐ and inter‐assay coefficients between 7.9% and 14.3%.     

A New 4.0-Generation Dendrimer Phosphorescence Labeling Reagent and Its Application to Determination of Trace Alkaline Phosphatase by Affinity Adsorption Solid Substrate-room Temperature Phosphorimetry

A Triton X-100-4.0G-D （4.0G-D refers to a 4.0-generation dendrimer） was brought forward as a new phosphorescence labeling reagent. Two types of specific affinity adsorption （AA） reactions （direct method and sandwich method） were carried out between the labeling product of Triton X-100-4：0G-D-Wheat germ agglutinin （WGA） and alkaline phosphatase （ALP）, the product of AA reaction preserved the good characteristics of room temperature phosphorescence （RTP） of 4.0G-D and △Ip of the product was proportional to the content of ALP. According to the fact stated above, a new method for the determination of trace ALP by affinity adsorption solid substrate-room temperature phosphorimetry （AA-SS-RTP） was established on the basis of WGA labeled with the Triton X-100-4.0G-D. The detection limits were 0.20 ag·spot^-1 （corresponding concentration： 5.0×10^-16 g·mL^-1, namely 5.0×10^-18 mol·L^-1） for a direct method and 0.14 ag·spot^-1 （corresponding concentration： 3.5×10^-16 g·mL^-1, namely 3.5×10^-18 mol·L^-1） for a sandwich method, respectively. For their high sensitivity, good repeatability and high accuracy, the direct method and sandwich method have been successfully appfied to determine the content of ALP in human serum, and the results were coincided with the clinical detection results of the enzyme-linked immunosorbent assay method by the Zhangzhou Hospital of Traditional Chinese Medicine. Meanwhile, the mechanism for the determination of trace ALP by AA-SS-RTP was discussed.     

Gold Nanoparticles Doped Three‐Dimensional Sol‐gel Matrix for Amperometric Human Chorionic Gonadotrophin Immunosensor

A novel electrochemical immunosensor for human chorionic gonadotrophin (hCG) was proposed by immobilization of hCG in gold nanoparticles doped three‐dimensional (3D) sol‐gel matrix and an interfacial competitive immunoreaction. The 3D organized composite structure was prepared by assemble of gold nanoparticles into a hydrolyzed (3‐mercaptopropyl)‐trimethoxysilane sol‐gel matrix, which showed good biocompatibility. After the interfacial competitive immunoreaction the formed HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive format, a method comprising of o‐phenylenediamine‐H₂O₂‐immobilized HRP labeled hCG immunoconjugate system for immunoassay of hCG from 5.0 to 30.0 mIU mL^?1 was developed. The immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility and could be used for detection of hCG in human serum with the consistent results in comparison with those obtained by a commercial analyzer.     

Synthesis of GoldMag particles with assembled structure and their applications in immunoassay

Micrometer-sized Fe3O4 particles and nano-sized gold particles were first synthesized by methods of self-aggregation of surface-chemically modified Fe3O4 nanoparticles and citrate reduction of the Au3 to Au0, respectively. Interaction between these two types of particles resulted in the assembly of nano-sized gold particles on the surface of the micrometer-sized Fe3O4 particles, forming an assembled structure with the Fe3O4 core particles around which are attached nano-sized gold parti- cles. The Fe3O4/Au structure is named GoldMag particles with assembled structure. The synthetic process, structure, and magnetic property of the GoldMag particles were analyzed. GoldMag particles with assembled structure have an irregular shape, rough surface with a diameter of 2―3 μm. These particles exhibit the superparamagnetic property with saturated magnetization of 41 A·m2/kg. In a single step, antibodies could be readily immobilized onto the surface of the particles with a high binding capacity. The GoldMag particles can be used as a novel carrier in immunoassays. The maximum quantity of human IgG immobilized onto GoldMag particles was 330 μg/mg. In order to validate the quality of the GoldMag particles as immunoassay carriers, an immunoassay system was used. The relative amount of immobilized human IgG was measured by HRP-labeled anti human IgG. The coefficient of variation within parallel samples of each group was below 6% and the coefficient of variation of means between five groups carried out separately was below 7%. Based on the sandwich method, the Hepatitis B surface antigen (HBsAg) and interleukin-8 (IL-8) were also analyzed by qualitative and quantitative detection, respectively. The result indicated that the GoldMag particles with assembled structure were an ideal carrier in immunoassay.     

Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification

An ultrasensitive electrogenerated chemiluminescence (ECL) immunoassay was proposed by using magnetic nanobeads (MNBs) as the carrier of ECL labels for ECL emission amplification. Carcinoembryonic antigen (CEA) and MNBs were initially immobilized on a platform in 1 : 1 molar ratio via sandwich immunoreaction. Subsequently, the MNBs were released from the platform and labeled with Ru(bpy)₃²⁺ species. After the MNBs with Ru(bpy)₃²⁺ were immobilized on an Au electrode, ECL of the Ru(bpy)₃²⁺ was measured for CEA determination. A linear relation between the ECL intensity and CEA concentration was obtained in a range of 1×10^?14 to 3×10^?13 mol/L (2.0 to 60 pg/mL) with a limit of detection of 8.0×10^?15 mol/L (1.6 pg/mL).     

A New Enzyme Immobilization Technique Based on Thionine‐Bovine Serum Albumin Conjugate and Gold Colloidal Nanoparticles for Reagentless Amperometric Biosensor Applications

A novel enzyme immobilization technique based on thionine‐bovine serum albumin conjugate (Th‐BSA) and gold colloidal nanoparticles (nano‐Au) was developed. Thionine was covalently bound onto the BSA film with glutaraldehyde(GA) as cross‐linker to achieve Th‐BSA conjugate. The free amino groups of thionine were then used to attach nano‐Au for the immobilization of horseradish peroxidase (HRP). Such nano‐Au/Th‐BSA matrix shows a favorable microenvironment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme. The proposed biosensor displays excellent catalytic activity and rapid response for H₂O₂. The linear range for the determination of H₂O₂ is from 4.9×10^?7 to 1.6×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelies‐Menten constant K value of 0.023 mM.     

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.     

Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus Plutonius

European foulbrood (EFB) is a honeybee larvae disease caused by a bacterium Melissococcus plutonius. An amperometric immunosensor based on a sandwich assay was developed for rapid point‐of‐care detection of this pathogen. An in‐house made anti‐Melissococcus antibody was immobilized to a gold surface of a screen‐printed sensor via self‐assembled monolayer of cysteamine activated with glutaraldehyde. The direct impedimetric detection of captured microbial cells was tested, however, a better performance was obtained after the formation of sandwich with the peroxidase‐labeled antibody in the amperometric mode. The label‐free assay was limited by higher non‐specific binding. The limit of detection of the immunosensor was 6.6×10⁴ CFU mL^?1 (colony‐forming units) with wide linear range between 10⁵ CFU mL^?1 and 10⁹ CFU mL^?1. The whole analysis was completed within 2 h, which is shorter compared to common laboratory diagnostic tools, such as enzyme‐linked immunosorbent assay or polymerase chain reaction. Furthermore, atomic force microscopy was used for confirmation of the bacteria presence on the electrode surface. The developed immunosensor was successfully employed in the analysis of real samples of honeybees and larvae. The achieved results demonstrate the potential of the amperometric immunosensor for practical in‐field diagnosis of EFB, which can prevent infection spreading and connected losses of honeybee colonies.     

Anodic‐Stripping Voltammetric Immunoassay for Ultrasensitive Detection of Low‐Abundance Proteins Using Quantum Dot Aggregated Hollow Microspheres

A new anodic‐stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer‐by‐layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti‐human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F_ab of IgG1 analyte from the sample. For detection, monoclonal anti‐human IgG1 (F_c‐specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich‐type immunoassay format, subsequent anodic‐stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL^?1 to 1.0 μg mL^?1 of IgG1 with a detection limit of 0.1 fg mL^?1. The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme‐linked immunosorbent assay method. The new immunoassay is promising for enzyme‐free, and cost‐effective analysis of low‐abundance biomarkers.     

Ultrasensitive Electrochemical Immunosensor for Detection of Tumor Necrosis Factor‐α Based on Functionalized MWCNT‐Gold Nanoparticle/Ionic Liquid Nanocomposite

A novel and highly sensitive electrochemical immunosensor was developed for the detection of protein biomarker tumor necrosis factor‐alpha (TNF‐α) based on immobilization of TNF‐α‐antibody (anti‐TNF‐α) onto robust nanocomposite containing gold nanoparticles (AuNP), multiwalled carbon nanotubes (MWCNTs) and ionic liquid (1‐buthyl‐3‐methylimidazolium bis (trifluoromethyl sulfonyl)imide). Functionalized MWCNT‐gold nanoparticle was produced by one‐step synthesis based on the direct redox reaction. The electrochemical properties of nanocomposite were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The anti‐TNF‐α was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti‐TNF‐α labeled with horseradish peroxidase as secondary antibody. Under optimum conditions, the immunosensor could detect TNF‐α in a linear range from 6.0 to 100 pg mL^?1 with a low detection limit of 2.0 pg mL^?1. The simple fabrication method, high sensitivity, good reproducibility, stability, as well as acceptable accuracy for TNF‐α detection in human serum samples are the main advantages of this immunosensor, which might have broad applications in protein diagnostics and bioassay.     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

Direct Electrochemistry of Glucose Oxidase Immobilized on Titanium Carbide‐Au Nanoparticles‐Fullerene C60 Composite Film and Its Biosensing for Glucose

The direct electrochemistry of glucose oxidase (GOD) immobilized on the designed titanium carbide‐Au nanoparticles‐fullerene C₆₀ composite film modified glassy carbon electrode (TiC‐AuNPs‐C₆₀/GCE) and its biosensing for glucose were investigated. UV‐visible and Fourier‐transform infrared spectra of the resulting GOD/TiC‐AuNPs‐C₆₀ composite film suggested that the immobilized GOD retained its original structure. The direct electron transfer behaviors of immobilized GOD at the GOD/TiC‐AuNPs‐C₆₀/GCE were investigated by cyclic voltammetry in which a pair of well‐defined, quasi‐reversible redox peaks with the formal potential (E^0′) of ‐0.484 V (vs. SCE) in phosphate buffer solution (0.05 M, pH 7.0) at the scan rate of 100 mV·s^?1 were obtained. The proposed GOD modified electrode exhibited an excellent electrocatalytic activity to the reduction of glucose, and the currents of glucose reduction peak were linearly related to glucose concentration in a wider linearity range from 5.0 × 10^?6 to 1.6 × 10^?4 M with a correlation coefficient of 0.9965 and a detection limit of 2.0 × 10^?6 M (S/N = 3). The sensitivity and the apparent Michaelis‐Menten constant (K_M^app) were determined to be 149.3 μA·mM^?1·cm^?2 and 6.2 × 10^?5 M, respectively. Thus, the protocol will have potential application in studying the electron transfer of enzyme and the design of novel electrochemical biosensors.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

Electrocatalysis via Direct Electrochemistry of Myoglobin Immobilized on Colloidal Gold Nanoparticles

The direct electron transfer between immobilized myoglobin (Mb) and colloidal gold modified carbon paste electrode was studied. The Mb immobilized on the colloidal gold nanoparticles displayed a pair of redox peaks in 0.1 M pH 7.0 PBS with a formal potential of –(0.108 ± 0.002) V (vs. NHE). The response showed a surface‐controlled electrode process with an electron transfer rate constant of (26.7 ± 3.7) s ^?1 at scan rates from 10 to 100 mV s^?1 and a diffusion‐controlled process involving the diffusion of proton at scan rates more than 100 mV s^?1. The immobilized Mb maintained its activity and could electrocatalyze the reduction of both hydrogen peroxide and nitrite. Thus, the novel renewable reagentless sensors for hydrogen peroxide and nitrite were developed, respectively. The activity of Mb with respect to the pseudo peroxidase with a K_M^app value of 0.65 mM could respond linearly to hydrogen peroxide concentration from 4.6 to 28 μM. The sensor exhibited a fast amperometric response to NO₂^? reduction and reached 93% of steady‐state current within 5 s. The linear range for NO₂^? determination was from 8.0 to 112 μM with a detection limit of 0.7 μM at 3σ.