Development of amperometric laccase biosensor through immobilizing enzyme in copper-containing ordered mesoporous carbon (Cu-OMC)/chitosan matrix

A functionalized copper-containing ordered mesoporous carbon (Cu-OMC) which shows good electrical properties was synthesized by carbonization of sucrose in the presence of cupric acetate inside SBA-15 mesoporous silica template. Based on this, a facilely fabricated amperometric biosensor by entrapping laccase into the Cu-OMC/chitosan (CS) film was developed. Laccase from Trametes versicolor was assembled on a composite film of Cu-OMC/chitosan (CS) modified Au electrode and the electrode was characterized. The optimum experimental conditions of biosensor for the detection of catechol were studied in details. Under the optimal conditions, the detection limit was 0.67 μM and the linear detection range was from 0.67 μM to 15.75 μM for catechol. The apparent Michaelis–Menten (K_M^app) was estimated using the Lineweaver–Burk equation and the K_M^app value was 40.2 μM. This work demonstrated that the Cu-OMC/CS composite provides a suitable support for laccase immobilization and construction of biosensor.     

Layer-by-layer assemblies of chitosan/multi-wall carbon nanotubes and glucose oxidase for amperometric glucose biosensor applications

A novel amperometric glucose biosensor based on multilayer films containing chitosan, multi-wall carbon nanotubes (MWCNTs) and glucose oxidase (GOD) was developed. MWCNTs were solubilized in chitosan (Chit-MWCNTs) used to interact with GOD. Poly (allylamine) (PAA) and polyvinylsulfuric acid potassium salt (PVS) were alternately deposited on the cleaned Pt electrode surface ((PVS/PAA)₃/Pt). The (PVS/PAA)₃/Pt electrode was alternately immersed in Chit-MWCNTs and GOD to assemble different layers of multilayer films. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Micrographs of MWCNTs were obtained by scanning electron microscope, and properties of the resulting biosensors were measured by electrochemical measurements. Among the resulting biosensors, the biosensor based on eight layers of multilayer films was best. The resulting biosensor was able to efficiently monitor glucose, with the response time within 8 s, a detection limit of 21 μM estimated at a signal-to-noise ratio of 3, a linear range of 1–10 mM, the sensitivity of 0.45 μA/mM, and well stability. The study can provide a feasible simple approach on developing a new immobilization matrix for biosensors and surface functionalization.     

Direct electrochemistry and electrocatalytic behavior of hemoglobin entrapped in chitosan/gold colloid/3-aminopropyl triethylene silane/Prussian blue composite film

Direct electrochemistry of hemoglobin (Hb) is achieved by immobilizing Hb-chitosan on a gold colloid/3-aminopropyl triethylene silane/Prussian blue composite film-modified glassy carbon electrode. The modified electrode exhibits a pair of well defined and quasi-reversible peaks with a formal potential of 0.205 V. The immobilized Hb retains its biological activity and shows high catalytic activity to the reduction of hydrogen peroxide. Experimental conditions influencing the biosensor performances such as pH and potential are optimized and assessed. Under the optimized conditions, the catalytic currents are linear to the concentrations of H₂O₂ in the ranges of 2-480 μM. The detection limit is 0.1 μM (S/N = 3). The electrochemical sensor has high stability and good reproducibility. Ultraviolet visible absorption spectra and Fourier transform infrared spectroscopy show that Hb keeps almost natural structure in the composite film. Therefore, the composite film is an alternative matrix for protein immobilization and biosensor preparation.     

Amperometric biosensor based on direct electrochemistry of hemoglobin in poly-allylamine (PAA) film

Hemoglobin (Hb) was immobilized in poly-allylamine (PAA) film onto the gold electrode by layer by layer (LBL) method. The modified electrode exhibited a pair of well-defined peaks during cyclic voltammetry, which was attributed from the direct electron transfer of heme proteins. The immobilized Hb showed an excellent electrocatalytical response to the reduction of hydrogen peroxide. The sensor exhibited a fast response and high sensitivity. Through the use of optimized conditions, the linear range for H₂O₂ detection was from 2.5 × 10^− 6 M to 5 × 10^− 4 M with detection limit of 0.2 μM. The proposed biosensor showed long-lasting stability and excellent reproducibility.     

Synthesis and enhanced electrochemical properties of pod-shaped gold/silica nanoparticles

Pod-shaped gold/silica nanoparticles (PGSNPs) were prepared using perfluorooctanoic acid (PFOA) and cetyltrimethylammonium bromide (CTAB) as cotemplates. The PGSNPs were utilized to explore a novel biosensor through coupling myoglobin (Mb) with chitosan (Chi). Compared with Mb-Chi-PSNPs (pod-shaped silica nanoparticles)/GC modified electrode, Mb-Chi-PGSNPs/GC electrode exhibited a pair of much stronger redox peaks at − 0.28 V (vs. Ag/AgCl). Moreover, facilitated direct electron transfer of the metalloenzymes with smaller peak-to-peak separation (ΔEp) of about 46 mV was acquired on the PGSNPs-based enzyme electrode. The PGSNPs-based biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with a wide linear range (1-540 µM) and high sensitivity (661 mA cm^− 2 M^− 1). Together, the Mb-Chi-PGSNPs film is one of ideal candidate materials for direct electrochemistry of redox proteins, and may find potential applications in biomedical, food, and environmental analysis and detection.     

Amperometric hydrogen peroxide biosensor based on cobalt ferrite-chitosan nanocomposite

A novel H₂O₂ biosensor based on horseradish peroxidase (HRP) immobilized into CoFe₂O₄-chitosan nanocomposite has been developed for the detection of hydrogen peroxide. The nanocomposite films were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). HRP has been entrapped into CoFe₂O₄-chitosan nanocomposite film and the immobilized enzyme could retain its bioactivity. This biosensor exhibited a fast amperometric response to hydrogen peroxide. The linear range for H₂O₂ determination was from 3 × 10^− 2 to 8 mM, with a detection limit of 2 × 10^− 3 mM based on S/N = 3. The response time of the biosensor was 4 s. The effects of the pH and the temperature of the immobilized HRP electrode were also studied.     

La_(0.7)Sr_(0.3)NiO_3/壳聚糖/聚吡咯/牛血红蛋白修饰玻碳电极的制备及构建过氧化氢传感器

在玻碳电极上修饰一层表面均匀的聚吡咯膜,然后利用钙钛矿和壳聚糖的复合膜来固定牛血红蛋白(Hb),制备出性能良好的过氧化氢生物传感器.采用循环伏安(CV)和扫描电镜对修饰电极进行了表征.该传感器对H2O2具有好的催化响应,且响应快.在优化的实验条件下,所制备的传感器对H2O2的线性范围为7.0×10-6～1.5×10-3mol/L,检测限为9.0×10-8mol/L.     

Carbon nanotube/chitosan/gold nanoparticles-based glucose biosensor prepared by a layer-by-layer technique

A new amperometric glucose biosensor was constructed, based on the immobilization of glucose oxidase (GOx) with cross-linking in the matrix of chitosan on a glassy carbon electrode, which was modified by layer-by-layer assembled carbon nanotube (CNT)/chitosan (CHIT)/gold nanoparticles (GNp) multilayer films. With the increasing of CNT/CHIT/GNp layers, the response current to H₂O₂ was changed regularly and the response current reached a maximum value when the number of CNT/CHIT/GNp layers was 8. The assembling process of multilayer films was simple to operate. With GOx as an enzyme model, a new glucose biosensor was fabricated. The excellent electocatalytic activity and special structure of the enzyme electrode resulted in good characteristics. The linear range was 6 × 10^? 6 ～ 5 × 10^? 3 M, with a detection limit of 3 × 10^? 6 M estimated at a signal-to-noise ratio of 3, fast response time (less than 6 s). Moreover, it exhibited good reproducibility and stability.     

Amperometric glucose biosensor based on in situ electropolymerized polyaniline/poly(acrylonitrile-co-acrylic acid) composite film

A new type of in situ electropolymerization was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous poly(acrylonitrile-co-acrylic acid)-coated platinum electrode in the presence of glucose oxidase. The results of EIS and SEM indicated the successful immobilization for enzyme in the composite polymer film. The novel glucose biosensor exhibited good selectivity and operational stability, which showed no apparent loss of activity after 40 consecutive measurements and intermittent usage for 45 days with storage in a phosphate buffer solution at 4°C. In addition, optimization of the biosensor construction as well as effects of applied potential, pH value of solution, temperature and common interfering compounds on the amperometric response of the sensor were investigated and discussed.     

Fabrication of amperometric xanthine biosensors based on direct chemistry of xanthine oxidase

The construction of amperometric xanthine biosensor by immobilization of xanthine oxidase (XOD) on the multi-wall carbon nanotubes (CNTs) modified glassy carbon (GC) electrode surface was investigated. The direct chemistry of XOD was accomplished and the formal potential was about − 0.465 V (vs SCE). The heterogeneous electron transfer rate constant was evaluated to be 2.0 ± 0.3 s^− 1. The xanthine biosensor based on XOD entrapped in silica sol–gel (SG) thin film on CNTs-modified GC electrode surface was also investigated. The XOD still maintains its activity to xanthine. The amperometric response to xanthine showed a linear relation in the range from 0.2 µM to 10 µM and a detection limit of 0.1 µM (S/N = 3). The enzyme electrode retained 95% of its initial activity after 90 days of storage. The sensor exhibited high sensitivity, rapid response and good long-term stability.     

酶-二维MXene复合材料的制备及其电化学检测H2O2的应用

本研究合成了具有垂直栅栏结构的二维MXene材料, 与辣根过氧化物酶进行固定, 构筑了过氧化氢电化学酶传感器。合成的MXene纳米栅栏具有大的比表面积, 优良的电子传导特性和在水溶液中的良好分散特性; 固定化在酶电极上的辣根过氧化物酶分子表现出了优良的过氧化氢催化效果。结果表明HRP@MXene/chitosan/GCE酶电化学传感器在过氧化氢浓度为5~1650 μmol/L范围内表现出很好的线性关系, 最低检测限为0.74 μmol/L, 且具有很好的操作稳定性, 该生物传感器被成功地应用于固态与液态食品中过氧化氢残留检测。     

Biopolymer-manganese oxide nanoflake nanocomposite films fabricated by electrostatic layer-by-layer assembly

Bio-nanocomposite films based on chitosan and manganese oxide nanoflake have been fabricated via the layer-by-layer (LBL) self-assembly technique. UV–vis absorption spectra showed that the subsequent growth of the nanocomposite film was regular and highly reproducible from layer to layer. X-ray photoelectron spectroscopy (XPS) spectra confirmed the incorporation of chitosan and manganese oxide nanoflake into the films. Scanning electron microscopy (SEM) images revealed that the nanocomposite film had a continuous surface and a layered structure. A sensitive hydrogen peroxide (H₂O₂) amperometric sensor was fabricated with the chitosan–manganese oxide nanoflake nanocompoite film. The sensor showed a rapid and linear response to H₂O₂ over the range from 2.5 × 10^? 6 to 1.05 × 10^? 3 M, with a sensitivity of 0.038 A M^? 1 cm^? 2.     

Spatial organization of peptide nanotubes for electrochemical devices

A novel biosensor for hydrogen peroxide was developed by combining the known properties of microperoxidase-11 (MP11) as an oxidation catalyst, and the interesting properties of diphenylalanine peptide nanotubes (PNTs) as a supporting matrix to allow a good bioelectrochemical interface. In this case, the synthesized MP11/PNTs were immobilized onto the ITO electrode surface via layer-by-layer (LBL) deposition, using poly(allylamine hydrochloride) (PAH) as positively charged polyelectrolyte layers. The PNTs provide a favorable microenvironment for MP11 to perform direct electron transfer to the electrode surface. The resulting electrodes showed a pair of well-defined redox peaks with formal potential at about −343 mV (versus SCE) in phosphate buffer solution (pH 7). The experimental results also demonstrated that the resulting biosensor exhibited good electrocatalytic activity to the reduction of H₂O₂ with a sensitivity of 9.43 μA cm⁻² mmol⁻¹ L, and a detection limit of 6 μmol L⁻¹ at the signal-to-noise ratio of 3. Moreover, we also observed that the peptides self-assembly can be influenced upon changing the pH of the solution. Alkaline solution appears to favor the packing of diphenylalanine nanotubes being closer than acidic or neutral conditions. The study proved that the combination of PNTs with MP11 is able to open new opportunities for the design of enzymatic biosensors with potential applications in practice.     

Preparation of RF sputtered AZO/Au thin film hydrogen peroxide sensitive electrode for utilization as a biosensor

In this study, hydrogen peroxide (H₂O₂) sensitive Al doped ZO(AZO)/Au thin film electrode has been developed for the utilization as a biosensor. A preferred c-axis oriented AZO/Au thin film was deposited on quartz substrate by RF magnetron sputtering at room temperature. Structural, morphological and optical properties of the AZO film were analyzed by X-ray diffraction, atomic force microscopy and photoluminescence. The sensor performance was characterized by electrochemical analysis device. The sensibility of prepared thin film electrodes to H₂O₂ was studied. The dependence of amperometric response current on the glucose and cholesterol concentrations was also investigated.     

A highly-sensitive l-lactate biosensor based on sol-gel film combined with multi-walled carbon nanotubes (MWCNTs) modified electrode

A new type of amperometric l-lactate biosensor based on silica sol-gel and multi-walled carbon nanotubes (MWCNTs) organic–inorganic hybrid composite material was developed. The sol-gel film was used to immobilize l-lactate oxidase on the surface of glassy carbon electrode (GCE). MWCNTs were used to increase the current response and improve the performance of biosensor. The sol-gel film fabrication process parameters such as H₂O : TEOS and pH were optimized, Effects of some experimental variables such as applied potential, temperature, and pH on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results showed that analytical performance of the biosensor could be improved greatly after introduction of the MWCNTs. Sensitivity, linear range, limit of detection (S / N = 3) were 2.097 μA mM^− 1, 0.3 to 1.5 mM, 0.8 × 10^− 3 mM for the biosensor without MWCNTs and 6.031 μA mM^− 1, 0.2 to 2.0 mM, 0.3 × 10^− 3 mM for the biosensor with MWCNTs, respectively. This method has been used to determine the l-lactate concentration in real human blood samples.     

Enzyme-immobilized Langmuir-Blodgett film for a biosensor

A lipid-protein monolayer for a biosensor was prepared utilizing a Langmuir- Blodgett technique. The enzyme glucose oxidase was used as the protein. Three types of lipid were chosen to change the surface charge of the polar group. The enzyme was immobilized on the lipid monolayer by adsorption from the subphase solution onto the lipid monolayer on the air/water interface. It was found that the lipid-enzyme interaction was dominated by electrostatic forces, and the characteristics of the film can be controlled by expansion and recompression of the adsorbed monolayer. Finally, a glucose sensor was fabricated by depositing the film onto a hydrogen peroxide electrode.     

Lipase immobilized on nanostructured cerium oxide thin film coated on transparent conducting oxide electrode for butyrin sensing

Nanostructured cerium oxide (CeO₂) thin films were deposited on transparent conducting oxide (TCO) substrate using spray pyrolysis technique with cerium nitrate salt, Ce(NO₃)₃·6H₂O as precursor. Fluorine doped cadmium oxide (CdO:F) thin film prepared using spray pyrolysis technique acts as the TCO film and hence the bare electrode. The structural, morphological and elemental characterizations of the films were carried out using X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray analysis (EDX) respectively. The diffraction peak positions in XRD confirmed the formation of highly crystalline ceria with cubic structure and FE-SEM images showed uniform adherent films with granular morphology. The band gaps of CeO₂ and TCO were found to be 3.2 eV and 2.6 eV respectively. Lipase enzyme was physisorbed on the surface of CeO₂/TCO film to form the lipase/nano-CeO₂/TCO bioelectrode. Sensing studies were carried out using cyclic voltammetry and amperometry, with lipase/nano-CeO₂/TCO as working electrode and tributyrin as substrate. The mediator-free biosensor with nanointerface exhibited excellent linearity (0.33–1.98 mM) with a lowest detection limit of 2 μM with sharp response time of 5 s and a shelf life of about 6 weeks.     

Amperometric choline biosensors based on multi-wall carbon nanotubes and layer-by-layer assembly of multilayer films composed of Poly(diallyldimethylammonium chloride) and choline oxidase

A layer-by-layer deposition technique combined with Multi-wall carbon nanotubes (MWCNTs) was employed for fabricating choline sensors. The terminals and side-walls were linked with oxygen-containing groups when MWCNTs were treated with concentrated acid mixtures. A film of MWCNTs was initially prepared on the platinum electrode surface. Based on the electrostatic interaction between positively charged polyallylamine (PAA) and negatively charged MWCNTs and poly(vinyl sulfate) (PVS), a polymer film of (PVS/PAA)₃ was alternately adsorbed on the modified electrode continuously to be used as a permselective layer. Then poly(diallyldimethylammonium) (PDDA) and choline oxidase(ChOx) multilayer films were assembled layer-by-layer on the pretreated electrode, so an amplified biosensor toward choline was constructed. The choline sensor showed a linear response range of 5 × 10^? 7 to 1 × 10^? 4 M with a detection limit of 2 × 10^? 7 M estimated at a signal-to-noise ratio of 3, and a sensitivity of 12.53 μA/mM with a response time of 7.6 s in the presence of MWCNTs. Moreover, it exhibited excellent reproducibility, long-term stability as well as good suppression of interference. This protocol could be used to immobilize other enzymes for biosensor fabrication.     

壳聚糖凝胶材料固定葡萄糖氧化酶制电极的研究

以壳聚糖为载体研究凝胶法固定葡萄糖氧化酶制电极。试验研究了载体壳聚糖的降解性；交联剂戊二醛的浓度、用量；电极的载酶量等固定化条件对所组建的传感器性能的影响。通过影响规律的分析、优化固定化条件的研究，找出了根据壳聚糖溶液粘度适当调整交联剂成二醛的用量和铂丝在酶膜母液中浸涂时间，克服壳聚糖的降解性对酶电极性能的影响，建立了制备性能相近的GOD传感器的方法。     

A novel potentiometric hydrogen peroxide sensor based on pKa changes of vinylphenylboronic acid membranes

A potentiometric hydrogen peroxide (H₂O₂) sensing scheme was developed using arylboronic acid as the electrode modifier. It is well-known that both aliphatic and aryl boronic acid undergo electrophilic displacement reaction with H₂O₂. This reaction involves replacement of boronic acid by the hydroxyl group of peroxide resulting in a change in pK_a value that can be exploited for sensing of H₂O₂. Vinylphenylboronic acid (VPBA) ink was prepared using Nafion as the binder and it was drop cast on an electrode surface. Morphology of the modified electrode was analysed using scanning electron microscopy (SEM). The present modifier exhibited a linear relationship between the difference in electrode potential (?Ep) vs. [H₂O₂] with a Nernstian slope of 26 ± 2 mV in the concentration range of 10^? 1–10^? 5 M. Application of the VPBA modified electrode for hydrogen peroxide sensing was studied in an industrial dye-bleach effluent.