Collagen‐Like Peptide as a Matrix for Enzyme Immobilization in Electrochemical Biosensors

An efficient enzyme immobilization technique is described in which well‐organized collagen peptide monolayers are used as a scaffold upon which horseradish peroxidase is anchored on gold electrode surfaces. The resulting electrode presents reproducible amperometric responses at an applied potential of ?0.1 V with a range of linearity for peroxides. These results suggest that triple helical collagen can be used for specific immobilization of HRP with advantages of low price, simplicity and biocompatibility. The results are significant for the control of biomolecular self‐assembly in the intrinsic electric devices.     

DNA-directed immobilization of horseradish peroxidase-DNA conjugates on microelectrode arrays: towards electrochemical screening of enzyme libraries

This work is aimed towards the generation of enzyme arrays on electrochemically active surfaces by taking advantage of the DNA-directed immobilization (DDI) technique. To this end, two different types of horseradish peroxidase (HRP)-DNA conjugates were prepared, either by covalent coupling with a bifunctional cross-linker or by the reconstitution of apo-HRP, that is, HRP lacking its prosthetic heme (protoporphyrin IX) group, with a covalently DNA-modified heme cofactor. Both conjugates were characterized in bulk and also subsequent to their immobilization on gold electrodes through specific DNA hybridization. Electrochemical measurements by using the phenolic mediator ortho-phenylendiamine indicated that, due to the high degree of conformational orientation, the apparent Michaelis-Menten constants of the reconstituted HRP conjugate were lower than those of the covalent conjugate. Due to the reversible nature of DDI, both conjugates could be readily removed from the electrode surface by simple washing and, subsequently, the electrodes could be reloaded with fresh enzymes, thereby restoring the initial amperometric-response activity. Moreover, the specific DNA hybridization allowed us to direct the two conjugates to distinct sites on a microelectrode array. Therefore, the self-assembly and regeneration capabilities of this approach should open the door to the generation of arrays of redox-enzyme devices for the screening of enzymes and their effectors.     

硫堇-牛血清白蛋白氧化还原复合膜修饰葡萄糖生物传感器的研究

通过交联法和自组装法制备了一种双酶型葡萄糖生物传感器.首先以牛血清白蛋白-戊二醛为交联剂以实现对辣根过氧化物酶(HRP)的固载,再利用凝集素-糖蛋白的识别作用将葡萄糖氧化酶(GOD)分子组装到电极表面,制得双酶型的葡萄糖生物传感器.采用原子力显微镜(AFM)考察了复合膜的性质,同时采用循环伏安法和计时电流法考察了该传感...     

Potential‐Assisted DNA Immobilization as a Prerequisite for Fast and Controlled Formation of DNA Monolayers

Highly reproducible and fast potential‐assisted immobilization of single‐stranded (ss)DNA on gold surfaces is achieved by applying a pulse‐type potential modulation. The desired DNA coverage can be obtained in a highly reproducible way within minutes. Understanding the underlying processes occurring during potential‐assisted ssDNA immobilization is crucial. We propose a model that considers the role of ions surrounding the DNA strands, the distance dependence of the applied potentials within the electrolyte solution, and most importantly the shift of the potential of zero charge during the immobilization due to the surface modification with DNA. The control of the surface coverage of ssDNA as well as the achieved speed and high reproducibility are seen as prerequisites for improved DNA‐based bioassays.     

Functionalized multiwall carbon nanotube/gold nanoparticle composites

Multiwall carbon nanotubes (MWCNTs) were chemically oxidized in a mixture of sulfuric acid and nitric acid (3:1) while being ultrasonicated. The effect of oxidative ultrasonication at room temperature on development of functional groups on the carbon nanotubes was investigated. The dispersability and the carboxylic acid group concentration of functionalized MWCNTs (fMWNTs) varied with reaction time. The concentration of carboxylic acid groups on fMWNTs increased from 4 x 10(-4) mol/g of fMWNTs to 1.1 x 10(-3) mol/g by doubling the treatment period from 4 to 8 h. The colloidal stability of aqueous fMWCNTs dispersions was enhanced through elongated oxidation. fMWCNTs that were reacted longer than 4 h did not precipitate in aqueous media for at least 24 h. The layer-by-layer self-assembly of polyelectrolytes on fMWCNTs was characterized by zeta potential measurements. The zeta potential of fMWCNTs changed from negative charge to positive charge when cationic polyelectrolytes were self-assembled on their surface. With addition of anionic polyelectrolytes, cationic polyelectrolyte coated fMWCNTs showed the expected charge reversal as expected for multilayer self-assembly. Complex formation of positively charged gold nanoparticles and negatively charged fMWCNTs was achieved with and without polyelectrolyte coatings by electrostatic interaction. The complex formation was characterized by high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy. The here found complex formation of positively charged colloidal gold and defect sites on fMWNTs indicates the location of functional groups on carbon nanotubes. It is suggested that positively charged colloids such as gold nanoparticles could be used for detection of defect sites on carbon nanotubes.     

Electrically Addressed Fabrication of Aptamer‐Based Array Electrodes

An aptamer immobilization method based electrically addressed fabrication has been developed for the preparation of aptamer‐modified arrayed electrodes, by which the human IgE aptamer was oriented and immobilized on the gold electrode surface. The optimization of the experimental conditions including the applied potential, time and scan rate of potential was investigated. The method was successfully used to immobilize the aptamer onto the desired electrodes, pixel by pixel, based on the electrically addressed approach. Compared to the control electrodes, the resulting aptamer‐modified electrodes showed their specific recognition for human IgE. The present method owns several advantages such as rapid and simple immobilization as well as its automatic addressed capability by the electric approach.     

Electrochemistry and electrocatalytic properties of mixed assemblies of horseradish peroxidase, poly(diallyl dimethylammonium chloride) and gold nanoparticles on a glassy carbon electrode

Poly(diallyldimethylammonium chloride), gold nanoparticles (Au-NPs; 2–5 nm) and horseradish peroxidase (HRP) were alternately self-assembled on a glassy carbon electrode. Due to the conducting wire effect of the Au-NPs, the HRP undergoes direct electrochemistry with a small peak-to-peak separation of 33 mV and a formal potential of ?0.370 V (versus SCE). The reaction involves a single electron transfer coupled to a one-proton transfer reaction. Electrochemical impedance spectroscopy and cyclic voltammetry also were applied to characterize the self-assembly process and to study the electrochemical behavior of the immobilized HRP. Its good electrocatalytic response to the reduction of hydrogen peroxide resulted in a novel third-generation biosensor for this species.     

Sunlight-induced covalent immobilization of proteins

Horseradish peroxidase (HRP) and glucose oxidase (GOD) were immobilized by sunlight onto the photoreactive cellulose membrane prepared by the reaction of cellulose membrane with 1-fluoro-2-nitro-4-azidobenzene (FNAB). A correlation between sunlight intensity and immobilization was studied. Sunlight intensity required for optimum immobilization was found to be 21,625 lux beyond which no appreciable increase in immobilization was observed. Around 2.5-fold increase in absorbance value was observed when HRP immobilization was carried out by sunlight than in dark or on untreated surface. Sunlight exposure gave better immobilization compared to 365 nm UV light. Thus, sunlight could be used as a potential alternative to UV light for immobilization of biomolecules such as carbohydrate, DNA or protein.     

New electroanalytical applications of self-assembled monolayers

New applications of self-assembled monolayers of thiol compounds on gold electrodes are reviewed. They include: (i) exploitation of electrical control of self-assembly of thiol compounds for electrically-addressable immobilization of receptor molecules onto sensor arrays; (ii) a spreader-bar technique for formation of stable nanostructures; and, (iii) use of self-assembled monolayers as selective filters for chemical sensors.     

Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

A new third‐generation biosensor for H₂O₂ assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Determination of carcinoembryonic antigen using a novel amperometric enzyme-electrode based on layer-by-layer assembly of gold nanoparticles and thionine

Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.     

Electrochemical studies of novel chitosan/TiO2 bioactive electrode for biosensing application

Bioactive electrode of uniformly dispersed TiO₂ in Chitoan (CS) was fabricated on ITO substrate for immobilization of horseradish peroxidase (HRP). Enhanced surface porosity and decrease in relative proportion of carbonyl functionality of CS in CS/TiO₂ matrix was observed. Current voltage characteristic of CS/TiO₂ matrix was enhanced by a factor of four possibly due to covalent and hydrogen binding of Ti atoms with hydroxyl and amino groups of CS. The immobilization of HRP on CS/TiO₂ had increased resistance for charge transfer. This is possibly due to strongly binding of HRP with CS/TiO₂ matrix and controlling transport of the ions of the supporting electrolyte.     

Gold adatom as a key structural component in self-assembled monolayers of organosulfur molecules on Au(1 1 1)

Chemisorption of organosulfur molecules, such as alkanethiols, arenethiols and disulfide compounds on gold surfaces and their subsequent self-organization is the archetypal process for molecular self-assembly on surfaces. Owing to their ease of preparation and high versatility, alkanethiol self-assembled monolayers (SAMs) have been widely studied for potential applications including surface functionalization, molecular motors, molecular electronics, and immobilization of biological molecules. Despite fundamental advances, the dissociative chemistry of the sulfur headgroup on gold leading to the formation of the sulfur–gold anchor bond has remained controversial. This review summarizes the recent progress in the understanding of the geometrical and electronic structure of the anchor bond. Particular attention is drawn to the involvement of gold adatoms at all stages of alkanethiol self-assembly, including the dissociation of the disulfide (S–S) and hydrogen-sulfide (S–H) bonds and subsequent formation of the self-assembled structure. Gold adatom chemistry is proposed here to be a unifying theme that explains various aspects of the alkanethiol self-assembly and reconciles experimental evidence provided by scanning probe microscopy and spectroscopic methods of surface science. While several features of alkanethiol self-assembly have yet to be revisited in light of the new adatom-based models, the successes of alkanethiol SAMs suggest that adatom-mediated surface chemistry may be a viable future approach for the construction of self-assembled monolayers involving molecules which do not contain sulfur.     

Immobilization of Horseradish Peroxidase on Nonwoven Polyester Fabric Coated with Chitosan

The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications.     

血清髓过氧化物酶电流型免疫传感器的研究

将Nafion吸附到玻碳电极表面,并通过静电吸附和共价键合作用将硫堇和纳米金层层自组装到Nafion修饰的电极表面,然后通过形成的纳米金单层吸附髓过氧化物酶(MPO)抗体,最后用辣根过氧化物酶(HRP)封闭电极上的非特异吸附位点,同时起到放大响应电流信号的作用,研制了一种检测MPO的新型电流型免疫传感器.实验结果表明,该免疫传感器对MPO的响应特性良好,其线性检测范围为2.5～100 μg/L; 检出限为1.425 μg/L,达到95%稳态响应时间<30 s,批间、批内的平均RSD(n=20) <2.94%和4.15%.电极的稳定性良好,在连续30 d内进行10次测量后,响应电流开始下降,平均测量值为初始的85.6%.探讨了抗体浓度、底物浓度、pH、温度及其它干扰物质等对该传感器的影响.使用本方法和经典的酶联免疫吸附实验(ELISA)同时对40份人血清标本MPO进行测试,结果表明: 两者相关性良好(r=0.9971, p<0.0001).该电流型免疫传感器具有灵敏度高、特异性好、不需标记和可以重复测量等优点.     

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.     

Fabrication of HRP/Bi_2WO_6 photoenzyme-coupled artificial catalytic system for efficiently degrading bisphenol A

A novel photoenzyme-coupled artificial catalytic system is fabricated by immobilizing horseradish peroxidase(HRP) on the Bi_2WO_6 hollow nanospheres via a facile electrostatic self-assembly process. The obtained Bi_2WO_6/HRP sample not only improves the visible light harvest ability but also promotes the high-efficiency separation of charge carriers. More importantly, the photogenerated electrons and produced H_2O_2 on Bi_2WO_6 directly take part in redox cycle reactions of HRP to induce photoenzyme synergic catalytic effect. In consequence, the degradation activity of Bi_2WO_6/HRP is significantly improved relative to Bi_2WO_6 and HRP for removing bisphenol A(BPA) under the visible light irradiation.This work launches a feasible design strategy for exploiting photoenzyme-coupled artificial catalytic system with special structure to degrade organic pollutants in water efficiently.     

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than ∼10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than ∼650 nM of anti-HSA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Hydrogen Peroxide Sensor Based on Horseradish Peroxidase Combined with CaCO3 Microspheres and Gold Nanoparticles

Direct electrochemistry and electrocatalysis of horseradish peroxidase(HRP) were achieved by entrapping the enzyme between CaCO3 microspheres and gold nanoparticles through forming sandwich configuration (CaCO3-HRP-AuNPs). Polyanion, poly(styrene sulfonate)(PSS), was hybrid with CaCO3 microspheres to increase the surface negative charges for binding with HRP through electrostatic interaction. After the bioconjugate CaCO3 PSS-HRP was entrapped in chitosan based sol-gel(CS-GPTMS) film, HRP was encapsulated by in situ formation of an outer layer of AuNPs through electrochemical reduction of HAuCl4. The composite film containing AuNPs, CaCO3-PSS-HRP bioconjugates and CS-GPTMS can provide favorable microenvironment for HRP to perform direct electron transfer at glassy carbon electrode(GCE). HRP retained its bioelectrocatalytic activity and lead to sensitive and fast amperometric response for the determination of H2O2. H2O2 could be detected in a very wide linear range from 5.0×10–6 mol/L to 7.1×10–2 mol/L. The sandwich configuration of CaCO3-biomolecules-AuNPs could serve as a versatile platform for enzyme immobilization and biosensing.