Biohybrid microarrays – Impedimetric biosensors with 3D in vitro tissues for toxicological and biomedical screening

To investigate the effectiveness of potential anti-cancer therapeutics or therapies, efficient screening methods are required. On the one hand, multicellular 3D aggregates (spheroids) are a powerful in vitro model for simulating the in vivo situation and on the other hand, planar electrode structures are generally highly suitable for automation and parallel testing. Here, the detection of the effect of active substances on spheroids positioned on electrodes of substrate integrated electrode arrays is exemplarily investigated. As a 3D tissue model a reaggregation system of T47D clone 11 tumor cells is used. The effect of cytotoxins (DMSO, Triton X-100) on spheroids can be detected by recording the effective impedance of planar electrodes covered by spheroids. The equivalent circuit model parameter of electrodes covered by cytotoxin treated spheroids are determined from recorded impedance spectra and compared to the parameter of electrodes covered by control spheroids as well as not covered electrodes. Spheroids on electrodes mainly influence the electrode impedance in the frequency range of 10 kHz to 1 MHz. The results are discussed in view of an optimal electrode/spheroid-interface for sensing the effects of therapeutics with high sensitivity.     

Comparison of Planar and 3‐D Interdigitated Electrodes as Electrochemical Impedance Biosensors

It has been reported that the introduction of a dielectric barrier between adjacent digits of an interdigitated electrode array can improve the sensitivity of the array as an electrochemical impedance biosensor. Here we present an in‐depth analysis of the impedance in planar interdigitated electrodes and 3‐D interdigitated electrodes (with dielectric barriers). The analysis indicates that the planar geometry not only provides lower impedance but also a higher change impedance as a result of molecular immobilization on the electrode array surface.     

Theory of Frequency-Dependent Polarization of General Planar Electrodes with Zeta Potentials of Arbitrary Magnitude in Ionic Media: 2. Applications and Results from Homogeneous and Array Systems of Electrodes

The expressions obtained in the previous paper for electrode polarization are applied to a homogeneous planar electrode and a planar array of electrodes used in the generation of nonuniform fields. The effective far field experienced outside the double layer is computed for both electrodes, and sample spectra are provided. The effective far field expression contains the electrode impedance and the effects of concentration polarization due to the static double layer on the electrode generated by the ζ potential. The effective far field results are compact and contain simple integrals that can be evaluated numerically.     

A new floating electrode structure for generating homogeneous electrical fields in microfluidic channels

In this article a new parallel electrode structure in a microfluidic channel is described that makes use of a floating electrode to get a homogeneous electrical field. Compared to existing parallel electrode structures, the new structure has an easier production process and there is no need for an electrical connection to both sides of the microfluidic chip. With the new chip design, polystyrene beads suspended in background electrolyte have been detected using electrical impedance measurements. The results of electrical impedance changes caused by beads passing the electrodes are compared with results in a similar planar electrode configuration. It is shown that in the new configuration the coefficient of variation of the impedance changes is lower compared to the planar configuration (0.39 versus 0.56) and less dependent on the position of the beads passage in the channel as a result of the homogeneous electrical field. To our knowledge this is the first time that a floating electrode is used for the realization of a parallel electrode structure. The proposed production method for parallel electrodes in microfluidic channels can easily be applied to other applications.     

Voltammetry at partially covered electrodes: Part III. Faradaic impedance measurements at model electrodes

Faradaic, impedances at model electrodes partially covered with a photoresist layer have been studied theoretically and experimentally. Equations for the faradaic impedance are derived based on the theoretical model and approach described in Part I of this series of papers. Experimental data for the hexacyanoferrate system at various model electrodes give excellent agreement, with theoretical predictions for the diffusion impedance behavior, and the applicability of the derived equations to the estimation of the degree of coverage and the size of the active regions is confirmed. Furthermore, the application of such model electrodes to the kinetic study of electrode reactions with high heterogeneous charge transfer rates is suggested.     

Effects of electrode size and surface morphology on electrode polarization in physiological buffers

Electrode polarization (EP) is inevitable in high conductivity buffers at low AC frequencies due to the accumulation of free charges at the electrode/electrolyte interface. Electrode miniaturization increases EP effect on impedance measurements. In this paper, six gold planar (GP) electrodes having different diameters () were used to investigate the size effect on EP with parallel plate electrode geometry. GP electrode surface was electrochemically deposited with gold nanostructures (GNs) to minimize the EP effect. Equivalent circuit model was used to attain electrode/electrolyte interfacial impedance. Constant phase element model was used to analyze the relation between the size and morphology of electrodes on EP. The surface morphology of gold nanostructured electrodes was examined using SEM, and the influence of different applied potential on the growth of GNs was elucidated with Nernst equilibrium condition. Surface roughness and wettability characteristics were examined performing surface roughness and contact angle measurements, respectively. The improvement of GNs deposited electrode performance was investigated by analytically generated Jurkat cell suspension spectra. The results show that the error in estimating the subcellular properties can be drastically reduced by using GNs deposited electrodes.     

Kinetics of electric double layer formation at the blocked spherical or cylindrical electrode/solid electrolyte interface under galvanodynamic and potentiodynamic conditions

The kinetics of charging of the blocked (inert) electrode/solid electrolyte interface is studied for spherical or cylindrical electrodes by the impedance method in two modes (galvanodynamic and potentiodynamic). The case of slow diffusion and adsorption-desorption is analyzed for species of one type, namely, defects of the solid-electrolyte rigid sublattice (minor carriers). The roles that both the slow lattice defects and the fast conduction ions play in the electric double layer formation are taken into account. Calculations involve the use of both the diffusion model of a spherical or cylindrical electrode in electrolyte (proposed by Jacobsen and West) and the ac circuit of an ideally polarizable planar electrode in a solid electrolyte (developed by Grafov, Ukshe, and Bukun).     

Hydrogel Microchambers Integrated with Organic Electrodes for Efficient Electrical Stimulation of Human iPSC‐Derived Cardiomyocytes

A hydrogel‐based microchamber with organic electrodes for efficient electrical stimulations of human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) is described. The microchamber is made from molecularly permeable, optically transparent, and electrically conductive polyvinyl alcohol (PVA) hydrogel and highly capacitive carbon electrode modified with poly(3,4‐ethylenedioxythiophene) (PEDOT). Spheroids of hiPSC‐CMs are cultured in microchambers, and electrically stimulated by the electrode for maturation. The large interfacial capacitance of the electrodes enables several days of electrical stimulation without generation of cytotoxic bubbles even when the electrodes are placed near the spheroids. The spheroids can be cultivated in the closed microchambers because of the permeated nutrients through the hydrogel, thus the spheroids are stably addressable and the culture medium around the sealed microchambers can be simply exchanged. Synchronized beating of the spheroids can be optically analyzed in situ, which makes it possible to selectively collect electrically responsive cells for further use. As the hydrogel is electrically conductive, the amount of electrical charge needed for maturing the spheroids can be reduced by configuring electrodes on the top and the bottom of the microchamber. The bioreactor will be useful for efficient production of matured hiPSC‐CMs for regenerative medicine and drug screening.     

Glass-based redox sensor

A new glass-based planar sensor for the determination of redox potentials is introduced. The sensor was fabricated by applying and sintering an iron-containing glass paste on an alumina substrate partially covered with a platinum layer. Regarding the condition of the functional glass layer, it was characterised using several physical and chemical methods. Furthermore, by means of the planar sensors, the determination of redox potentials of different redox-active substances was carried out. It could be shown that the measurement results obtained with these electrodes were comparable to those received with conventional noble metal-based redox electrodes. For sensors prepared by selected glass compositions, a significant pH dependence of the measurement signal was not detected. Moreover, the electrode could be used as working electrode for cyclic voltammetry, too.     

Continuous-time random-walk theory of interfering diffusion and chemical reaction with an application to electrochemical impedance spectra of oxidized Zr-1%Nb

A microscopic theory is developed for the interplay of diffusion and chemical reaction and the results are compared with electrode impedance measurements on an oxide electrode. The theory is based on the ideas of continuous-time random walk and accounts for the interference of diffusion and recombination of the charge carriers in the oxide. The treatment results in a dispersive diffusivity with two time constants, one of them corresponding to the random walk, the other to the reaction. Combining this diffusivity with the Warburg electrode admittance expression, which refers to cases where the rate-limiting step is diffusion in a semi-infinite medium bounded by a plane, an admittance function is obtained. The phase angle is found to be higher than 45 degrees distinguishing it from the Gerischer impedance which was developed for a related problem. The oxides were produced by hydrothermal oxidation of Zr-l%Nb alloy, a metal used as cladding material for nuclear fuel elements. The electrode impedance spectra of Zr/Zr-oxide electrodes in aqueous SO(3) (2-) solutions were taken at various anodic voltages between 1 Hz and 100 kHz and temperatures between 278 and 333 K. The theoretical admittance functions could be successfully compared with the observed spectra. Both the functional forms and the fitted parameter values support our theory which is also in keeping with Macdonald's point-defect model.     

Drug testing on 3D in vitro tissues trapped on a microcavity chip

Close to realistic responses to anti-cancer drugs are not adequately provided in monolayer or single cells assays. 3-dimensional multicellular cultures (spheroids) mimicking in vivo-like conditions are established as cell biological models for microtumors/metastases. For a non-invasive real-time monitoring of the electrical parameters of such spheroid cultures we designed, fabricated and tested a 3D multifunctional electrode-based microcavity array. In a non-adherent assay acute tests with tumor spheroids were done maintaining their spherical shape and cellular arrangement. The sensor chip with 15 individual square microcavities containing four gold electrodes each was used for impedance spectroscopy to analyze the tissue models in terms of morphological and structural changes. Cell type specific differences in the spectra and varying responses to several anti-tumor drugs were found. Further development of the prototype will provide a promising tool for the use in pharmacological high-throughput studies.     

Fundamentals of lead-acid cells: Part XII. The reduction of lead sulphate

The reduction of lead sulphate grown anodically on planar and porous electrodes has been studied by a potentiostatic step technique. Both “planar” and “porous” electrodes behave in a similar manner and indicate that the electrode reduction reaction has a finite depth of penetration into the electrode. The kinetics of the formation of metallic lead on both electrodes appear to be instantaneous nucleation and two-dimensional growth processes with subsequent current limitations due to overlap and lead sulphate depletion. The current limitation processes are very complex and it has not been found possible to fit a satisfactory mathematical model.     

宏观均相多孔电极电化学阻抗谱基础

电化学阻抗谱可用于诊断多孔电极内电荷转移反应,即界面电荷集聚和电荷传导,以及反应物质输运。本文采用复相量方法,在同态假设条件下,重新推演多孔电极阻抗谱模型,厘清传统多孔电极阻抗谱模型中的模糊性表述。(1) 定义多孔电极表征输入参数,包括电极基体电子电导率σ₁ 、电解质离子电导率σ₂、界面电荷传递电导率g_ct、单位面积界面电容C、固相扩散系数D、速度常数k、电极厚度d、特征孔深L_p 和单位体积表面积S_c;(2) 解析阻抗谱特征输出参数,包括场扩散常数K,特征频率ω₀、ω₁、ω₂、ω₃和 ω_max,它们分别相关于界面传导反应、有限场扩散、氧化还原反应、孔内扩散和最小特征孔尺寸,以及分别对应于从传导到扩散和从扩散到饱和的转折频率f_k1 和f_k2;(3) 当参数X和Z同时变化时(X = σ₁和Z = d,S_c,L_p,C,g_ct,D,k),通过阻抗谱特征参数的演变规律,分析了电荷转移反应中X和Ζ参数耦合竞争;(4)为深入分析电荷转移反应中参数X和Z的耦合竞争,引入了分叉频率f_XZ和f_ZX 。f_XZ和f_ZX所处位置可以用于表征参数X和Z影响电荷转移反应的深度和广度。当分叉频率f_XZ和f_ZX不存在时,表明电荷转移反应中参数X和Z在全频率范围内存在耦合竞争。总之,借助于特征频率和分叉频率,本文一方面研究了动力学参数和微观结构参数对多孔电极中电荷转移反应的影响,另一方面分析谱图的变化及其背后的阻抗谱特征演化规律。本文研究结果可为阻抗谱的系统仿真和辨识提供理论基础,可为多孔电极内电荷转移反应的竞争分析提供技术支撑,还可为电化学储能系统的优化设计提供诊断工具。     

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.     

The impedance of sintered plate cadmium electrodes in alkaline solution

It has been shown that the a.c. impedance of a sintered plate cadmium electrode can be related to that for a planar cadmium electrode by considering the penetration of the a.c. signal down the pores of the sinter matrix. Evenly and poorly-impregnated electrodes were studied and complex impedance plane plots obtained for regions corresponding to double layer charging, active dissolution and passivation. At high frequencies when porous behaviour was observed, the results displayed a close agreement with those predicted by the simple a.c. theory developed for a porous electrode with semi-infinite pores. Discrepancies could be attributed to the presence of uncharged Cd(OH)₂ or Cd(OH)₂ precipitate formed during discharge. The effect of charge-discharge cycling was investigated and the resulting impedance spectra interpreted in terms of changes in the wetted areas of cadmium, redistribution of active material and the progressive build up of Cd(OH)₂ within the sinter pores.     

A novel strategy for fabrication,activation and cleaning of fully 3D printed flexible planar electrochemical platforms

In recent years, 3D printing of carbon-based conductive filaments has received growing attention for assembling electrodes to be used in a wide variety of electroanalytical devices and applications. Despite the large amount of work present in literature concerning the development of three-dimensional (3D) conductive structures, its potential as dry deposition method for assembling two-dimensional (2D) electrodes to be used in planar configuration is still largely unexplored. In fact, the possibility to rapidly change the geometry of the electrochemical circuits, associated with the reduction of waste and the absence of solvents, which are instead important components of ink and paste formulations, makes this strategy a valid green and efficient alternative to other deposition approaches such as screen-printing technology. We report here a rapid and solvent-free method for assembling fully 3D printed flexible planar electroanalytical platforms (3DEPs) to be used with microliters of liquid. At the same time, a novel protocol for the surface pre-treatment of 3D printed electrodes based on ultrasonication in aqueous NaOH solution followed by electrochemical activation using the same medium, is presented. In addition, the same procedure has proved to be efficient for cleaning the electrode surface after electrochemical passivation, thus confirming the validity of both time-efficient and environmentally-friendly assembling and activation/cleaning procedures developed which allow efficient and reusable electrodes to be produced. Finally, 3DEPs were tested by a proof-of-concept quantification of a commonly used food dye (Brilliant Blue, E-133) in commercial solutions used for homemade food coloring.     

Voltammetry at partially covered electrodes: Part I. Chronopotentiometry and chronoamperometry at model electrodes

Equations for chronopotentiometry and chronoamperometry at partially covered electrodes have been derived using a model of hexagonal array of cylidrical spaces terminated, at the electrode surface, by concentric active and inactive regions. The boundary value problem was shown to be analogous to that for a charge transfer preceded by a chemical reaction. Experiments with the reduction of ferricyanide on gold model electrodes partially covered with photoresist layer showed excellent agreement with the theory. Application of the equations to estimation of coverage and size of active sites distributed on a electrode surface is discussed.     

Electrochemical Impedance Analysis of Thermogalvanic Cells

Thermogalvanic cells(also known as thermo-electrochemical cells) that convert waste heat energy to electricity are a new type of energy conversion device. However, the electron transfer kinetics and mass transfer of redox couples have not been thoroughly studied. Here, the ion reaction and charge transport in thermogalvanic cells are investigated by electrochemical impedance analysis. We first propose the detailed impedance model followed experimental verification on three types of electrode materials. Parameters including kinetic rate constants and ion diffusion coefficients for the electrodes are obtained by fitting the impedance data. Our study shows explicitly that impedance analysis can provide useful information on selecting suitable electrode materials for thermogalvanic cells.     

An electrode array for electrochemical immuno-sensing using the example of impedimetric tenascin C detection

Electrochemical biosensors allow simple, fast and sensitive analyte detection for various analytical problems. Especially immunosensors are favourable due to specificity and affinity of antigen recognition by the associated antibody. We present a novel electrode array qualified for parallel analysis and increased sample throughput. The chip has nine independent sample chambers. Each chamber contains a circular gold working electrode with a diameter of 1.9 mm that is surrounded by a ring-shaped auxiliary electrode with a platinum surface. The corresponding silver/silver chloride reference electrodes are embedded in a sealing lid. The chip is open to the full range of electrochemical real-time detection methods. Among these techniques, impedance spectroscopy is an attractive tool to detect fast and label-free interfacial changes originating from the biorecognition event at the electrode surface. The capabilities of the novel electrode array are demonstrated using the example of tumour marker tenascin C detection. This glycoprotein of the extracellular matrix is expressed in cancerous tissues, especially in solid tumours such as glioma or breast carcinoma. Electrodes covered with specific antibodies were exposed to tenascin C containing samples. Non-occupied binding sites were identified using a secondary peroxidase-conjugated antibody that generated an insoluble precipitate on the electrode in a subsequent amplification procedure. The charge transfer resistance obtained from impedimetric analysis of ferri-/ferrocyanide conversion at the electrode served as analytic parameter. This assay detected 14 ng (48 fmol) tenascin C that is sufficient for clinical diagnostics. The electrode surface could be regenerated at least 20-fold without loss of its analytical performance.     

3-D electrode designs for flow-through dielectrophoretic systems

Traditional methods of dielectrophoretic separation using planar microelectrodes have a common problem: the dielectrophoretic force, which is proportional to nabla|E|2, rapidly decays as the distance from the electrodes increases. Recent advances in carbon microelectromechanical systems have allowed researchers to create carbon 3-D structures with relative ease. These developments have opened up new possibilities in the fabrication of complex 3-D shapes. In this paper, the use of 3-D electrode designs for high-throughput dielectrophoretic separation/concentration/filtration systems is investigated. 3-D electrode designs are beneficial because (i) they provide a method of extending the electric field within the fluid. (ii) The 3-D electrodes can be designed so that the velocity field coincides with the electric field distribution. (iii) Novel electrode designs, not based on planar electrodes designs, can be developed and used. The electric field distribution and velocity fields of 3-D electrode designs that are simple extensions of 2-D designs are presented, and two novel electrode designs that are not based on 2-D electrode designs are introduced. Finally, a proof-of-concept experimental device for extraction of nanofibrous carbon from canola oil is demonstrated.