Direct Electrochemistry of Hemoglobin Immobilized on Carbon‐Coated Iron Nanoparticles for Amperometric Detection of Hydrogen Peroxide

A novel method for preparation of hydrogen peroxide biosensor was presented based on immobilization of hemoglobin (Hb) on carbon‐coated iron nanoparticles (CIN). CIN was firstly dispersed in a chitosan solution and cast onto a glassy carbon electrode to form a CIN/chitosan composite film modified electrode. Hb was then immobilized onto the composite film with the cross‐linking of glutaraldehyde. The immobilized Hb displayed a pair of stable and quasireversible redox peaks and excellent electrocatalytic reduction of hydrogen peroxide (H₂O₂), which leading to an unmediated biosensor for H₂O₂. The electrocatalytic response exhibited a linear dependence on H₂O₂ concentration in a wide range from 3.1 μM to 4.0 mM with a detection limit of 1.2 μM (S/N=3). The designed biosensor exhibited acceptable stability, long‐term life and good reproducibility.     

Trans-membrane electron transfer in red blood cells immobilized in a chitosan film on a glassy carbon electrode

We have studied the trans-membrane electron transfer in human red blood cells (RBCs) immobilized in a chitosan film on a glassy carbon electrode (GCE). Electron transfer results from the presence of hemoglobin (Hb) in the RBCs. The electron transfer rate (k _s) of Hb in RBCs is 0.42 s^?1, and <1.13 s^?1 for Hb directly immobilized in the chitosan film. Only Hb molecules in RBCs that are closest to the plasma membrane and the surface of the electrode can undergo electron transfer to the electrode. The immobilized RBCs displayed sensitive electrocatalytic response to oxygen and hydrogen peroxide. It is believed that this cellular biosensor is of potential significance in studies on the physiological status of RBCs based on observing their electron transfer on the modified electrode.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

银-磷酸钛纳米复合膜的构建及其电化学生物传感

利用ITO基底上层层组装构建的多层内嵌银纳米粒子的磷酸钛薄膜固定了血红蛋白并且用于生物传感研究。由于银纳米粒子与磷酸钛膜的协同作用,实验中可以观察到Hb的直接电子传递。研究表明所制备的Hb-Ag-TiP/PDDA/ITO电极对H₂O₂响应迅速、稳定,检测限达3.3×10^-6 mol·L^-1。     

Direct electrochemistry and electrocatalysis of hemoglobin in a multilayer {nanogold/PDDA}n inorganic–organic hybrid film

A new amperometric biosensor for hydrogen peroxide (H₂O₂) has been developed that is based on direct electrochemistry and electrocatalysis of hemoglobin (Hb) in a multilayer inorganic–organic hybrid film. o-Phenylenediamine (PDA) was electropolymerized onto a glassy carbon electrode (GCE), and then negatively charged nanogold particles and positively charged poly(diallyldimethylammonium chloride) (PDDA) were alternately assembled on the PDA/GCE surface. Finally, Hb was electrostatically adsorbed on the surface of gold nanoparticles. The electrochemical behavior of the resulting biosensor (Hb/{nanogold/PDDA}_n/PDA/GCE) was assessed and optimized. The performance and factors influencing the biosensor were studied in detail. Under optimal conditions, the immobilized Hb displayed good electrocatalytic response to the H₂O₂ reduction ranging from 1.3 μM to 1.4 mM with a detection limit of 0.8 μM (at 3δ). In addition, the biosensor exhibited rapid response, good reproducibility, and long-term stability. Electronic supplementary material to this paper is available in electronic form at Correspondence: Dianyong Tang, Department of Chemistry and Life Science, Leshan Teachers College, Sichuan (Leshan) 614000, P.R. China     

A hydrogen peroxide biosensor based on Langmuir-Blodgett technique: Direct electron transfer of hemoglobin in octadecylamine layer

The present paper describes the modification of hemoglobin (Hb)-octadecylamine (ODA) Langmuir-Blodgett (LB) film on a gold electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide. Atomic force microscopy (AFM) image of Hb-ODA LB film indicated Hb molecules existed in ODA layer in a well-ordered and compact form. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of 4.58 ± 0.95 s⁻¹ and a formal potential of −185 mV (versus Ag/AgCl) in phosphate buffer (1.0 mM, pH 5.0) contain 0.1 M KCl at a scan rate of 200 mV s⁻¹, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The formal potential of Hb heme Fe(III)/Fe(II) redox couple in ODA film shifted linearly between pH 5 and 8 with a slope of −23.8 mV pH⁻¹, suggesting that proton took part in electrochemical reaction. The ODA could accelerate the electron transfer between Hb and the electrode. This modified electrode showed an electrochemical activity to the reduction of hydrogen peroxide (H₂O₂) without the aid of any electron mediator.     

A gold electrode modified with hemoglobin and the chitosan@Fe3O4 nanocomposite particles for direct electrochemistry of hydrogen peroxide

We report on a novel electrochemical biosensor that was fabricated by immobilizing hemoglobin (Hb) onto the surface of a gold electrode modified with a chitosan@Fe₃O₄ nano-composite. The Fe₃O₄ nanoparticles were prepared by co-precipitation and have an average size of 25 nm. They were dispersed in chitosan solution to obtain the chitosan@Fe₃O₄ nano-composite particles with an average diameter of 35 nm as verified by transmission electron microscopy. X-ray diffraction patterns and Fourier transform IR spectroscopy confirmed that the crystallite structure of the Fe₃O₄ particles in the nano-composite has remained unchanged. At pH 7.0, Hb gives a pair of redox peaks with a potential of about ?0.21 V and ?0.36 V. The Hb on the film maintained its biological activity and displays good electrocatalytic reduction activity towards hydrogen peroxide. The linear range for the determination of H₂O₂ is from 2.3 μM to 9.6 mM, with a detection limit at 1.1 μM concentration (at S/N?=?3). The apparent Michaelis-Menten constant is 3.7 mM and indicates the high affinity of Hb for H₂O₂. This biosensor also exhibits good reproducibility and long-term stability. Thus, it is expected to possess potential applications in the development of the third-generation electrochemical biosensors.

Immobilization of hemoglobin on SBA-15 applied to the electrocatalytic reduction of H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The direct electron transfer between hemoglobin (Hb) and an electrode was realized by first immobilizing the protein onto SBA-15.The results of the immobilization showed that the adsorption was pH-dependent with a maximum adsorption near the isoelectric point of the protein, and SBA-15 with a larger pore diameter showed greater adsorption capacity for Hb. UV–vis spectroscopy and nitrogen adsorption analysis indicated that Hb was adsorbed within the channel of SBA-15 and no significant denaturation occurred to the protein. The Hb/SBA-15 composite obtained was used for the fabrication of a Hb biosensor to detect hydrogen peroxide. A pair of well-defined redox peaks at −0.337 and −0.370 V on the Hb/SBA-15 composite modified glassy carbon electrode was observed, and the electrode reactions showed a surface-controlled process with a single proton transfer at a scan rate range from 20 to 1,000 mV/s. The sensor showed a fast amperometric response, a low detection limit (2.3 × 10⁻⁹ M) and good stability for the detection of H₂O₂. The electrochemical results indicated that the immobilized Hb still retained its biological activity.     

Amperometric hydrogen peroxide biosensor based on the immobilization of heme proteins on gold nanoparticles-bacteria cellulose nanofibers nanocomposite

A novel matrix, gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au-BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au-BC nanocomposite which was proved by UV-vis and IR spectra. The response of the developed biosensor to H₂O₂ was related to the amount of AuNPs in Au-BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1 s) to H₂O₂, a good linear response over a wide range of concentration from 0.3 μM to 1.00 mM, and a low detection limit of 0.1 μM based on S/N = 3. The high performance of the biosensor made Au-BC nanocomposite superior to other materials as immobilization matrix.     

Novel Hydrogen Peroxide Biosensor Based on Hemoglobin Combined with Electrospinning Composite Nanofibers

A facile strategy to construct an amperometric biosensor was described for the determination of hydrogen peroxide (H₂O₂). This biosensor relied on an electrospinning gold nanoparticle-chitosan-poly(vinyl alcohol) composite nanofibers modified ITO electrode, followed by immobilization of hemoglobin (Hb) on the surface. The introduction of nanofibers and gold nanoparticles in the modification of electrode surface not only enhanced the surface area of the modified electrode for enzyme immobilization but also facilitated the electron transfer rate. Under optimum conditions, the sensor was characterized in terms of its morphology by scanning electron microscopy and its electroactivity by cyclic voltammetry and chronoamperometry. Scanning electron microscopy revealed that the obtained nanofibers were uniform. The chronoamperometric behavior of the modified electrode indicated that the immobilized Hb retained electrochemical activity inside the electrospinning fibrous membranes. The electrode responded linearly to H₂O₂ in a wider concentration range of 5.6 × 10^?7 M to 5.2 × 10^?2 M with a low detection limit (S/N = 3) of 1.98 × 10^?7 M and a short response time of ～4 s, suggesting a much better performance than that of other sensors. Moreover, the biosensor achieved bulk production and exhibited superior properties for the sensitive determination of H₂O₂, studied namely, long-term stability, good reproducibility, and high selectivity.     

Amperometric Biosensors Based on Platinum Nanowires

Abstract

Platinum nanowires (PtNW) were prepared by an electrodeposition strategy using nanopore alumina template. The nanowires prepared were dispersed in chitosan (CHIT) solution and stably immobilized onto the surface of glassy carbon electrode (GCE). The electrochemical behavior of PtNW‐modified electrode and its application to the electrocatalytic reduction of hydrogen peroxide (H₂O₂) are investigated. The modified electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. As an application example, the glucose oxidase was immobilized onto the surface of PtNW‐modified electrode through cross‐linking by glutaric dialdehyde. The detection of glucose was performed in phosphate buffer at –0.2 V. The resulting glucose biosensor exhibited a short response time (<8 s), with a linear range of 10^?5?10^?2 M and detection limit of 5×10^?6 M.     

Electrochemical Surface Structuring with Polyaniline Wrapped Hb for Hydrogen Peroxide Biosensing

Through the electrodeposition of aniline with hemoglobin (Hb) on zincoxide‐gold colloidal sols (ZnO‐AuNPs) modified indium oxide electrode, a hydrogen peroxide (H₂O₂) biosensor was constructed. Polyaniline (PANI) form a nano‐cage wrapped Hb, which provided a comfortable and stable site for the immobization of Hb. UV‐vis spectrum was employed to characterize Hb retained original structure in the resulting Hb‐PANI/ZnO‐AuNPs membrane. Electrochemical investigation of the biosensor showed a pair of well‐defined, quasi‐reversible redox peaks with E_pa= ‐0.139 V and E_pc = ‐0.238 V (vs. SCE) in 0.1 M pH 7.0 phosphate buffer solution at the scan rate of 100 mV/s. The biosensor displayed a fast response time (<3 s) and broad linear response to H₂O₂ in the range from 1.5 μM to 1.7 mM with a detection limit of 0.8 μM (S/N = 3).     

基于壳聚糖-纳米金/纳米普鲁士蓝/L-半胱氨酸修饰的 葡萄糖传感器的研究

在金电极表面修饰一层L-半胱氨酸,再利用静电吸附作用固定纳米普鲁士蓝(nano-PB),然后利用壳聚糖-纳米金复合膜将葡萄糖氧化酶(GOD)固定于修饰电极表面,制成新型的葡萄糖传感器.通过交流阻抗技术,循环伏安法和计时电流法考察了电极的电化学特性.在优化的实验条件下,该传感器在葡萄糖浓度为3.0×10-6～1.0×10-3 mol/L范围内有线性响应,检测下限为1.6×10-6 mol/L.此外该传感器具有响应快、稳定性好和选择性良好的特点,能有效排除常见干扰物质如抗坏血酸、尿酸等对测定的影响.     

A study of uricase biosensor based on a glassy carbon electrode modified with Nafion and methyl viologen

A uricase biosensor was constructed by using bovine serum albumin and glutaraldehyde as cross linker to immobilize uricase on a glassy carbon electrode modified with Nafion and methyl viologen (MV). A linear response of uric acid in serum was observed in a range of 1.0·10^–3 to 5.0·10^–6 mol/1 and the response time is 25 s. This biosensor has the advantage of high sensitivity, fast response as compared with previous sensors and low interferences.     

Hydrogen peroxide sensor based on horseradish peroxidase immobilized on a silver nanoparticles/cysteamine/gold electrode

A third-generation hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) on a gold electrode modified with silver nanoparticles. A freshly-cleaned gold electrode was first immersed in a cysteamine–ethanol solution, and then silver nanoparticles were immobilized on the cysteamine monolayer, and finally HRP was adsorbed onto the surfaces of the silver nanoparticles. This self-assemble process was examined via atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited an excellent electrocatalytic response toward the reduction of hydrogen peroxide. The linear range of the biosensor was 3.3 M to 9.4 mM, and the detection limit was estimated to be 0.78 M. Moreover, the biosensor exhibited a fast response, high sensitivity, good reproducibility, and long-term stability.     

Direct electrochemistry of hemoglobin on a room temperature ionic liquid modified electrode and its electrocatalytic activity for the reduction of oxygen

A room temperature ionic liquid (RTIL), 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIm][BF₄]), was successfully immobilized on the surface of a basal plane graphite (BPG) electrode through silica sol and Nafion film to form a sol/RTIL/Nafion modified electrode. Direct electrochemistry of hemoglobin (Hb), which was adsorbed on the surface of sol/RTIL/Nafion modified electrode, was investigated. The results from cyclic voltammetry (CV) suggested that Hb could be tightly adsorbed on the surface of the electrode. A couple of well-defined and quasi-reversible CV peaks of Hb can be observed in a phosphate buffer solution (pH 7.0). RTIL shows an obvious promotion for the direct electro-transfer between Hb and electrode. Hb adsorbed on electrode surface exhibits an obvious electrocatalytic activity for the reduction of oxygen O₂. The reduction peak currents were proportional linearly to the concentration of oxygen in the range 0.14–1.82 μM. A third generation biosensor based on RTIL can be constructed for the determination of O₂.     

Electrochemical characteristics of a platinum electrode modified with a matrix of polyvinyl butyral and colloidal Ag containing immobilized horseradish peroxidase

A new hydrogen peroxide biosensor was constructed, which consisted of a platinum electrode modified by a matrix of polyvinyl butyral (PVB) and nanometer-sized Ag colloid containing immobilized horseradish peroxidase (HRP), and using Co(bpy)₃³⁺ as mediator in the hydrogen peroxide solution. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The HRP immobilized on colloidal Ag was stable and retained its biological activity. The sensor displays excellent electrocatalytic response to the reduction of H₂O₂. Analytical parameters such as pH and temperature were also studied. Linear calibration for H₂O₂ was obtained in the range of 1×10^–5 to 1×10^–2 M under optimized conditions. The sensor was highly sensitive to H₂O₂, with a detection limit of 2×10^–6 M, and the sensor achieved 95% of steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability.     

The Modification of Screen‐Printed Carbon Electrodes with Amino Group and Its Application to Construct a H2O2 Biosensor

Electrooxidation of thionine on screen‐printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen‐printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H₂O₂ biosensor exhibited a fast response (2 s) and low detection limit (0.5 μM).     

基于聚亚甲基蓝-碳纳米管复合膜固载血红蛋白的H2O2生物传感器

采用吸附和电化学聚合修饰方法,制得了聚亚甲基蓝-碳纳米管聚合膜玻碳电极(PMB-MWNTs/GCE),再将血红蛋白(Hb)固定在PMB-MwNTs/GCE表面,制备了稳定的Hb/PMB-MwNTs//GCE的H2O2生物传感器,并用循环伏安法对修饰电极的生物电催化行为进行了表征.研究结果表明,固定在PMB-MWNTs/...     

Colloid Au-enhanced DNA immobilization for the electrochemical detection of sequence-specific DNA

We use colloidal Au to enhance the DNA immobilization amount on a gold electrode and ultimately lower the detection limit of our electrochemical DNA biosensor. Self-assembly of approximately 16-nm diameter colloidal Au onto a cysteamine modified gold electrode resulted in an easier attachment of an oligonucleotide with a mercaptohexyl group at the 5′-phosphate end, and therefore an increased capacity for nucleic acid detection. Quantitative results showed that the surface densities of oligonucleotides on the Au colloid modified gold electrode were approximately (1–4)×10¹⁴ molecules cm⁻². Hybridization was induced by exposure of the ssDNA-containing gold electrode to ferrocenecarboxaldehyde labeled complementary ssDNA in solution. The detection limit is 5×10⁻¹⁰ mol l⁻¹ of complementary ssDNA, which is much lower than our previous electrochemical DNA biosensors. The Au nanoparticle films on the Au electrode provide a novel means for ssDNA immobilization and sequence-specific DNA detection.