Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors,DNA‐Sensors,and Enzyme Biosensors

Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial‐functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field‐effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen‐antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single‐base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field‐effect transistors (FET) upon the formation of antigen‐antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme‐based biosensors. The reconstitution of apo‐enzymes on cofactor‐functionalized electrodes and the formation of cofactor‐enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article.     

生物功能电极 III. 葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合, 将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响, 并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现, 由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性, 酶反应表观上遵循Michealis-Menten动力学。     

生物功能电极 Ⅲ.葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合,将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响,并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现,由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性,酶反应表观上遵循Michealis-Menten动力学。     

Bioelectrochemical studies on catalase modified glassy carbon paste electrodes

The enzyme catalase (EC: 1.11.1.6) has been covalently coupled onto the surface of glassy carbon (GC) powder matrix using a 16 atom spacer arm. The enzyme coupled powder was made into a paste electrode that was used to study the electrochemical properties. Standard electrochemical techniques like cyclic voltammetry, differential pulse voltammetry and flow injection analysis studies were carried out using this paste electrode. The cyclic voltammogram of the modified paste exhibited a clear increase in the reduction peak at −180 mV in the presence of hydrogen peroxide. The potential at which maximum Faradaic activity was observed was determined using differential pulse voltammetry, which showed a clear peak at −100 mV. This potential was used to monitor the response of the electrode to varying substrate concentrations using a home made setup for flow injection analysis. A linear increase in the current values in the range 0.1–1 mM hydrogen peroxide concentration was observed in our system.     

Disposable Gold Electrode Array for Simultaneous Electrochemical Studies

An efficient and inexpensive eight gold electrode array has been manufactured by a combination of screen printing and gold electrodeposition techniques. Gold electrodeposition was performed in potentiostatic and galvanostatic conditions. Different treatments, involving temperature and polishing control, led to electrodes with different roughness. The electrochemical behavior of the generated gold surface was studied by cyclic voltammetry showing the characteristic response of polycrystalline gold, in contrast with disposable gold electrodes fabricated by screen printing from gold inks. The electrodes were chemically modified through the adsorption of alkanethiols self‐assembled monolayers and the coupling of a model protein. Both reactions were followed by cyclic voltammetry and Electrochemical Impedance Spectroscopy (EIS). The electrodes have shown high reproducibility in their electrochemical behavior as well as in their modifications.     

Direct Electron Transfer at a Glucose Oxidase–Chitosan-Modified Vulcan Carbon Paste Electrode for Electrochemical Biosensing of Glucose

This article describes the investigation of direct electron transfer (DET) between glucose oxidase (GOD) and the electrode materials in an enzyme-catalyzed reaction for the development of improved bioelectrocatalytic system. The GOD pedestal electrochemical reaction takes place by means of DET in a tailored Vulcan carbon paste electrode surfaces with GOD and chitosan (CS), allowing efficient electron transfer between the electrode and enzyme. The key understanding of the stability, biocatalytic activity, selectivity, and redox properties of these enzyme-based glucose biosensors is studied without using any reagents, and the properties are characterized using electrochemical techniques like cyclic voltammogram, amperometry, and electrochemical impedance spectroscopy. Furthermore, the interaction between the enzyme and the electrode surface is studied using ultraviolet–visible (UV–Vis) and Fourier transform infrared (FTIR) spectroscopy. The present glucose biosensor exhibited better linearity, limit of detection (LOD?=?0.37?±?0.02 mol/L) and a Michaelis–Menten constant of 0.40?±?0.01 mol/L. The proposed enzyme electrode exhibited excellent sensitivity, selectivity, reproducibility, and stability. This provides a simple “reagent-less” approach and efficient platform for the direct electrochemistry of GOD and developing novel bioelectrocatalytic systems.     

Interaction of DNA with echinomycin at the mercury electrode surface as detected by impedance and chronopotentiometric measurements

The capacitance measurement (dependence of the differential capacitance C of the electrode double layer on potential E, C-E curves), electrochemical impedance spectroscopy (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dt/dE on potential at constant current, chronopotentiometric stripping analysis-CPSA) have been used for electrochemical study of echinomycin and its interaction with single-stranded (ss) and double-stranded (ds) DNA at the hanging mercury drop electrode (HMDE). The capacitance measurement showed that echinomycin gives a pseudocapacitance redox peak strongly dependent on the a.c. voltage frequency at the potential of -0.53 V. This peak is observed with dsDNA-echinomycin complex as well, but not with ssDNA treated by echinomycin. Similar results were obtained using CPSA measurements. Thus capacitance measurements and CPSA can distinguish with the aid of the bis-intercalator echinomycin the single-stranded and double helical form of DNA adsorbed at the mercury electrode surface. Impedance measurement in connection with adsorptive transfer technique can find the differences between ssDNA and dsDNA, which promise to use this technique for detection of dsDNA in hybridisation reactions.     

A comprehensive analysis of impedance of the electrochemical cell

A comprehensive impedance characteristics of two electrodes electrochemical cell has been presented. In this method a multisinusoidal current excitation signal is used. The change of potential of both the electrodes are all registered as a function of time. The proposed method gives the possibility of determining the impedance of both electrodes individually as well as the impedance of a two-electrode system. Additional application of short time Fourier transform of time registers allows the determination of changes in the measured impedance values over time. In order to present the possibilities of the proposed technology it was applied to a process of charging commercially available electrochemical cell NiCd. The new measurement methodology allows understanding the dynamics of processes occurring in a electrochemical cell. This is the basis for the development of effective and affordable electro-catalysts. Thanks to results obtained with Dynamic Electrochemical Impedance spectroscopy (DEIS) method it is possible to understand the mechanisms and kinetics of processes occurring in electrochemical cells while charging.     

Design of Micro‐interdigitated Electrodes and Detailed Impedance Data Analysis for Label‐free Biomarker Quantification

Electrical impedance based biosensing is a label‐free technique that is gaining momentum in biology/medicine. The electrical impedance, typically measured using an array of micro‐fabricated interdigitated electrode array (IDE), is a byproduct of the interaction between electric fields and target bio‐molecules/cells. In current impedance based biosensing, it has been focused on utilizing the magnitude of the impedance (|Z|) to detect/quantify bio‐molecules. There were no reports on designing IDE electrodes, sensitivity analysis and detailed impedance data analysis. To address this issue, we have designed and fabricated IDE array and performed model experiments. We have found that depending on the frequency of the external electric potential, there is a variation of electric field across the array of IDEs from first pair to last pair. We then developed impedance data analysis technique (using (|Z|) and its phase (φ)) to analyze the complex impedance data, and finally, we have utilized Warburg theoretical circuit model to calculate the capacitance and resistance of the individual IDE pairs in the constant phase impedance region. Using the capacitance values, we have developed a procedure to determine the sensitivity of the IDE array. We have found that sensitivity of the IDE array does not depend on the sample conductivity.     

Impedimetric characterization of the changes produced in the electrode–solution interface by bacterial attachment

This paper describes a new method for measuring the attachment of bacteria, specifically Escherichia coli on platinum electrodes using impedance spectroscopy. Impedance spectroscopy measurements showed that the double layer capacitance of the electrode was very sensitive both to the concentration of bacteria in the solution and to the attachment time. Impedance measurements of E. coli were compared with classical measurements of bacterial attachment on identical electrodes such as staining/microscopy and bacterial removal by sonication and plating onto agar. The relationship between the measured impedance of the electrode during attachment and the biophysical processes involved is discussed.     

Ordered Mesoporous Silica Incorporating Platinum Nanoparticles as Electrode Material for Paracetamol Detection

High ordered mesoporous materials (SBA-15) modified with Al and/or B and Pt nanoparticles (Pt NPs) were used for preparing modified graphite paste electrodes (Pt/M−SBA-15-GPE, where M=Al−, B− or Al−B−) and applied for paracetamol (PA) detection. The electrodes were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Square wave voltammetry (SWV) technique was used to obtain the analytical parameters for PA detection. The acquired values of electrochemical and analytical parameters recommend the mesoporous compound containing Pt NPs to be used as composite electrode material for PA detection in real samples.     

Electrochemical behavior of nickel in electrolytes based on 1-n-butyl-3-methylimidazolium tetrafluoroborate ionic liquid for capacitor applications

Electrochemical studies were performed using Ni electrodes in solutions of a mixture of ethylene glycol or of γ-butyrolactone with 1-n-butyl-3-methylimidazolium tetrafluoroborate ionic liquid. The aim of the study was to evaluate the use of these systems in electrochemical double-layer capacitor. Cyclic voltammetry experiments showed a potential range at which the Ni electrode behaved as a polarizable electrode. Ni oxidizes at high anodic potentials. Inside the potential range without electrochemical activity, the capacitance and the solution resistance, which were evaluated by impedance electrochemical spectroscopy, were compared for the two solutions tested. Conductivity measurements of the electrolytes with different compositions were also acquired. The results of cyclic voltammetry indicated that the Ni has a wide electrochemical window and low current peak densities of oxidation in the γ-butyrolactone medium than in ethylene glycol medium. The γ-butyrolactone and 1-n-butyl-3-methylimidazolium tetrafluoroborate ionic liquid solutions had the highest conductivity values. Decreased 1-n-butyl-3-methylimidazolium tetrafluoroborate ionic liquid content in different solvent mixtures resulted in an increase in the capacitance value at the Ni/electrolyte interface. The highest capacitance values were obtained for Ni in ethylene glycol and 1-n-butyl-3-methylimidazolium tetrafluoroborate medium.     

Electrochemical Characterization of Carbon Solidlike Paste Electrode Assembled Using Different Carbon Nanoparticles

Solid like carbon paste electrodes (SCPEs) are built using different carbon materials namely carbon black N110, N220, N375, N772 and acetylene black. The electrochemical behavior of these electrodes and the influence of carbon black/paraffin ratio were studied and the results were discussed and compared to other electrodes prepared with graphite, mesoporous carbon and nanopowder carbon. Cyclic voltammetry, amperometry and electrochemical impedance spectroscopy were employed for their electrochemical and analytical characterizations. Amperometric measurements using N110, N220, N375 SCPEs with solid paraffin, showed a linear response of benzoquinone concentration with a detection limit of 75, 32 and 171 nM respectively.     

The impedance related to the electrochemical hydrogen insertion into WO3 films – On the applicability of the diffusion-trapping model

Impedance analysis of electrochemically prepared WO₃ films has been carried out in order to investigate the applicability of the diffusion-trapping model to the analysis of impedance spectra related to electrochemical hydrogen insertion. The impedance spectra measured under different conditions have been analyzed by using CNLS fitting. It has been shown, that the expressions derived for the diffusion-trapping model adequately describe the impedance response of the system. Despite of the great number of adjustable parameters many of them could be determined with a good statistics, and reasonable estimated mean values have been obtained for the others. The reliability of the estimated parameters was checked by comparing the results with existing experimental data.     

Ordered Mesoporous Carbon Paste Electrodes for Electrochemical Sensing and Biosensing

Surface renewable ordered mesoporous carbon paste electrodes (OMCPE) were prepared by mechanical mixing ordered mesoporous carbon (OMC) and mineral oil. Electrochemical behavior of the composite electrode was evaluated and compared with the conventional graphite paste (GPE) and carbon nanotubes paste (CNTPE) electrodes. The OMCPE provided improved electron transfer kinetics and catalytic capabilities in connection with oxidation and/or reduction of different redox systems, such as ferricyanide and some biological species, e. g. ascorbic acid (AA), uric acid (UA), β‐nicotinamide adenine dinucleotide (NADH), dopamine (DA), epinephrine (EP), acetaminophenol (AP) and hydrogen peroxide. The substantial decrease in the over voltage of the hydrogen peroxide oxidation along with the facile incorporation of glucose oxidase (GOD) into the composite matrix allowed us successfully to fabricate a sensitive and selective glucose biosensor. A linear response up to 15 mM glucose was obtained for the OMCPE modified with 10% GOD (w/w) with a detection limit of 0.072 mM. In addition, we also successfully applied the OMCPE to the anodic stripping voltammetric analysis of heavy metal ions with improved sensitivities in comparison with CNTPE and GPE. The excellent experimental results implicate that the new developed paste electrode holds great promise in the design of electrochemical devices, such as sensors and biosensors.     

Amperometric biosensors for glucose based on carbon paste modified electrodes

The modification of carbon-paste electrodes by incorporation of the enzyme glucose oxidase (GOD) is described. The resulting probes can be operated as amperometric glucose sensors in the presence or absence of a mediator (1,1'-dimethylferrocene) mixed into the paste. Extended linear calibration ranges have been obtained up to 90 and 5OmM glucose respectively. The electrode responses were rapid, reaching steady-state values within 30-40 sec. Advantages of using a GOD-paste formulation are suggested. Plasma glucose assays were correlated with spectrophotometric determinations based on glucose oxidase (y = 1.07x - 0.16, r = 0.973, n = 17).     

一种新的测定微粒的电化学技术

设计了一种由2个石墨电极短路相连组成工作电极的新的电化学池装置.操作时首先通过外力按压使极少量固体微粒粘附在其中一个石墨电极表面上,然后在溶液存在下将微粒夹紧并固定在2个石墨电极表面之间进行电化学测定.电化学转化过程中生成的可溶性物质被封闭在2个石墨电极表面之间而得到测定.用该技术对钯沉积在氧化铝上而组成的催化剂的电化学行为以及黄铁矿的电化学行为进行了研究.结果表明,其兼具可电解粘合剂碳糊电极和固体微粒伏安法(voltammetry of microparticles)技术的优点而避免了各自的缺点:即不使用粘合剂,从而消除了粘合剂中杂质产生的氧化或还原电流的影响;可测定电化学转化过程中生成的可溶性物质;分辨率好、易于操作.     

Carbon film electrodes for oxidase-based enzyme sensors in food analysis

Carbon film resistor electrodes have been evaluated as transducers for the development of multiple oxidase-based enzyme electrode biosensors. The resistor electrodes were first modified with Prussian Blue (PB) and then covered by a layer of covalently immobilized enzyme. Electrochemical impedance spectroscopy was used to characterize the interfacial behaviour of the Prussian Blue modified and enzyme electrodes; the spectra demonstrated that the access of the substrates is essentially unaltered by application of the enzyme layer. These enzyme electrodes were used to detect the substrate of the oxidase (glucose, ethanol, lactate, glutamate) via reduction of hydrogen peroxide at +50 mV versus Ag/AgCl in the low micromolar range. Response times were 1-2 min. Finally, the glucose, ethanol and lactate electrochemical biosensors were used to analyse complex food matrices—must, wine and yoghurt. Data obtained by the single standard addition method were compared with a spectrophotometric reference method and showed good correlation, indicating that the electrodes are suitable for food analysis.     

Chitin and chitin-cellulose composite hydrogels prepared by ionic liquid-based process as the novel electrolytes for electrochemical capacitors

This paper reports on the preparation and electrochemical performance of chitin- and chitin-cellulose-based hydrogel electrolytes. The materials were prepared by a casting solution technique using ionic liquid-based solvents. The method of chitin dissolution in ionic liquid with the assistance of dimethyl sulfoxide co-solvent was investigated. The obtained membranes were soaked with 1-M lithium sulfate aqueous solution. The prepared materials were preliminarily characterized in terms of structural and physicochemical properties. Further, the most promising biopolymer membranes were assembled with activated carbon cloth electrodes in symmetric electrochemical capacitor cells. The electrochemical performances of these devices were studied in a 2-electrode system by commonly known electrochemical techniques, such as cyclic voltammetry, galvanostatic charge/discharge, and electrochemical impedance spectroscopy. The devices operated at a maximum voltage of 0.8 V. All the investigated materials have shown high efficiency in terms of specific capacitance, power density, and cyclability. The studied capacitors exhibited specific capacitance values in the range of 92–98 F g⁻¹, with excellent capacitance retention (ca. 97–98%) after 20,000 galvanostatic charge and discharge cycles. Taking into account the above information and the eco-friendly nature of the biopolymer, it appears that the prepared chitin- and chitin-cellulose-based hydrogel electrolytes can be promising components for green electrochemical capacitors.

     

季铵盐掺杂聚苯胺电极的电容性能

采用循环伏安法,在铂电极表面聚合制备了季铵盐[CnH2n+1N(CH3)3]Cl(n=12,14,16,18)掺杂的聚苯胺修饰电极。 利用扫描电子显微镜、红外光谱以及X射线衍射对复合电极的表面形貌和结构进行了表征。 用循环伏安法、交流阻抗和恒电流充放电测试对电极的电化学性质和电容行为进行了系统研究。 结果表明,其中[C18H37N(CH3)3]Cl季铵盐掺杂的聚苯胺复合电极比表面积大,电容性能好,在2×10-3 A的充电电流下,初始比电容高达329.6 F/g,未掺杂电极比电容为199.0 F/g。 而且,复合电极的循环稳定性良好,经30次循环后比电容保持为252.4 F/g。