CMOS-Based Bio/Chemosensor and Bioelectronic Microsystems

Microfabrication techniques and, in particular, complementary-metal-oxide-semiconductor (CMOS) technology have been used to devise chemo/biosensors [1-3] as well as bioelectronic microsystems [4-7]. Examples of micromachined bio- or chemosensors, such as cantilevers or micoelectrode arrays, will be shown, and the electrical interfacing of CMOS microelectronics with biological entities or electrogenic cells, i.e., cells that react upon electrical stimulation and, in turn, produce electrical signals (heart cells or neurons) are detailed. CMOS-based, fully integrated microelectrode arrays for bidirectional communication (stimulation and recording) with electrogenic cells are presented. These devices are capable of monitoring relevant electrophysiological responses of cells to electrical stimuli or to pharmacological agents with prospective applications in the field of bio-inspired information processing or pharmascreening.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.     

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer’s disease, or vision impairment caused by ganglion cell degeneration in the retina.     

基于一维和二维纳米材料的神经界面构筑

Neural interfaces have contributed significantly to our understanding of brain functions as well as the development of neural prosthetics. An ideal neural interface should create a seamless and reliable link between the nervous system and external electronics for long periods of time. Implantable electronics that are capable of recording and stimulating neuronal activities have been widely applied for the study of neural circuits or the treatment of neurodegenerative diseases. However, the relatively large cross-sectional footprints of conventional electronics can cause acute tissue damage during implantation. In addition, the mechanical mismatch between conventional rigid electronics and soft brain tissue has been shown to induce chronic tissue inflammatory responses, leading to signal degradation during long-term studies. Thus, it is essential to develop new strategies to overcome these existing challenges and construct more stable neural interfaces. Owing to their unique physical and chemical properties, one-dimensional (1D) and two-dimensional (2D) nanomaterials constitute promising candidates for next-generation neural interfaces. In particular, novel electronics based on 1D and 2D nanomaterials, including carbon nanotubes (CNTs), silicon nanowires (SiNWs), and graphene (GR), have been demonstrated for neural interfaces with improved performance. This review discusses recent developments in neural interfaces enabled by 1D and 2D nanomaterials and their electronics. The ability of CNTs to promote neuronal growth and electrical activity has been proven, demonstrating the feasibility of using CNTs as conducting layers or as modifying layers for electronics. Owing to their good mechanical, electrical and biological properties, CNTs-based electronics have been demonstrated for neural recording and stimulation, neurotransmitter detection, and controlled drug release. Different from CNTs-based electronics, SiNWs-based field effect transistors (FETs) and microelectrode arrays have been successfully demonstrated for intracellular recording of action potentials through penetration into neural cells. Significantly, SiNWs FETs can detect neural activity at the level of individual axons and dendrites with a high signal-to-noise ratio. Their ability to record multiplexed intracellular signals renders SiNWs-based electronics superior to traditional intracellular recording techniques such as patch-clamp recording. Besides, SiNWs have been explored for optically controlled nongenetic neuromodulation due to their tunable electrical and optical properties. As the star of the 2D nanomaterials family, GR has been applied as biomimetic substrates for neural regeneration. Transparent GR-based electronics combining electrophysiological measurements, optogenetics, two-photon microscopy with multicellular calcium imaging have been applied for the construction of multimodal neural interfaces. Finally, we provide an overview of the challenges and future perspectives of nanomaterial-based neural interfaces.     

Biosensor on the base of polypyrrole modified ultramicroelectrode arrays

A novel approach for the construction of a micro biosensor on the base of amperometric enzyme microelectrode arrays is described in this paper. The technique contains the electrochemical deposition of different enzymes in a conducting organic polymer, e.g. polypyrrole, on a transducer built up with the help of microfabrication technology. The electrochemical characteristics of these microelectrode arrays can be compared to conventional microelectrodes with the advantage of higher current outputs. For the first time different model enzymes like glucose oxidase, choline oxidase and lactate oxidase have been tested showing the principal possibility to construct a micro biosensor on the base of ultramicroelectrode arrays.     

Cell adhesion promotion strategies for signal transduction enhancement in microelectrode array in vitro electrophysiology: An introductory overview and critical discussion

Microelectrode arrays (MEAs) find application both in vitro and in vivo to record and stimulate electrical activity in electrogenic cells such as neurons, cardiomyocytes, pancreatic beta cells or immortalized cell lines derived therefrom (e.g., PC12, HL-1). In MEA electrophysiology, the quality of the predominantly extracellularly recorded or elicited electrical signals strongly depends on the distance, strength and stability of the interfacial contact between the electrogenic cells and an electrode. Decorating the substrate or electrode with biochemical adhesion factors and physical guidance cues does not only determine the tightness of that junction, but it also modulates substrate biocompatibility, its biostability, cell differentiation as well as cell fate. If an interface is furthermore topologically, chemically or physically patterned or constrained, neural interconnectivity may be steered towards directional organization. In this introductory and selective overview, we briefly discuss adhesion events at the chemical and biological level, review the general role and mechanisms of cell adhesion in (neuro)biology, then explore how cells adhere to artificial substrates. This will lead to the discussion of popular strategies for enhancing and steering interfacial interactions at the bio-hardware boundary with particular focus on MEA substrates. It will include a critical treatment of open issues with respect to the origin and shape of extracellularly recorded signals and their modulation by cell-culture-inherent events.     

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.     

Enhanced optical immunosensor based on surface plasmon resonance for determination of transferrin

Wavelength modulation surface plasmon resonance biosensors (SPR) using colloidal Au nanoparticles and double-linker sensing membrane enhancement are reported for determination of transferrin. The 2-mercaptoethylamine (MEA) was immobilized on the biosensor surface with traditional amine coupling method. The interaction between colloidal Au nanoparticles and MEA was investigated. The anti-transferrin was immobilized on the biosensor surface prepared with staphylococcal protein A (SPA). The interaction of the antibody and antigen was monitored in real time. The good response was obtained in the concentration range 1-20, 0.1-20 and 0.05-20 μg/mL for directly immune assay, double-linker assay and colloidal Au-amplified assay. The result clearly demonstrates that these methods may obtain significantly enhancement of sensitivity for the wavelength modulation SPR biosensor.     

Spatio-temporally resolved measurement of quantal exocytosis from single cells using microelectrode array modified with poly L-lysine and poly dopamine

The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array(MEA), fabricated by MEMS technology, was applied successfully for real-time monitoring of quantal exocytosis from single pheochromocytoma(PC12) cell.The developed MEA was evaluated by dopamine(DA) using electrochemical methods and the results revealed that the sensitivity of DA was improved to 12659.24 μA L mmol ~(-1)cm~(-2). The modified MEA was used to detect in vitro vesicular exocytosis of DA from single PC12 cells stimulated by concentrated100 mmol L~(-1)K~+cell solution. A total of 592 spikes were measured and analyzed by three parameters and the statistical results revealed the population of each parameter was an approximate Gaussian distribution, and on average, 1.31×10~6 ±9.25×10~4 oxidizable molecules were released in each quantal exocytosis. In addition, results also indicate that a single PC12 cell probably releases the spikes with T ranging from 25.6 ms to 35.4 ms corresponding to I_(max)ranging from 45.6 pA to 65.2 pA. The devices, including a homemade computer interface and the MEA modified with polymer film, provides a new means for further research on the neural, intercellular, communication mechanism.     

In vitro continuous amperometric monitoring of 5-hydroxytryptamine release from enterochromaffin cells of the guinea pig ileum

A diamond microelectrode was used to sensitively, reproducibly and stably record overflow of 5-hydroxytryptamine (5-HT, serotonin) from enterochromaffin cells (EC) of the intenstinal mucosal layer. 5-HT is an important neurotransmitter and paracrine signalling molecule in the gastrointestinal tract. The diamond microelectrode was formed by overcoating a sharpened 76 microm diameter Pt wire with a thin layer of conducting diamond. After insulation with polypropylene, the conically-shaped microelectrode had a diameter of about 10 microm at the tip and 80 microm at the cylindrical portion. The exposed length was 100-200 microm. Continuous amperometry with the microelectrode poised at a detection potential of 700 mV vs. Ag|AgCl was used to measure 5-HT overflow as an oxidation current. 5-HT overflow was elicited by both mechanical and electrical stimulation. Some minor electrode fouling, a common problem with the oxidative detection of 5-HT, was seen for diamond but the response stabilized enabling recording in vitro. Both 5-HT and the paracrine hormone, melatonin, were detected in the extracellular solution. The 5-HT oxidation current increased in the presence of the serotonin transporter (SERT) inhibitor, fluoxetine (1 microM), providing evidence that the oxidation current was associated with 5-HT.     

基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法

基于微流体脉冲驱动控制技术搭建了电化学微流控芯片的制备系统.首先将纳米银墨水和甘油溶液分别微喷射到玻璃基底表面形成微电极图形和微流道液体阳模图形;然后分别进行烧结和聚二甲基硅氧烷(PDMS)模塑工艺制得微电极和微流道;最后将微电极和微流道键合形成电化学微流控芯片.研究了系统参量对液滴产生的影响以及液滴直径和重叠率对液滴成线的影响,制得的微电极最小线宽为45 μm、厚度为2.2 μm、电阻率为5.2 μΩ·cm,制得的微流道最小线宽为35 μm,流道表面光滑.采用制得的电化学微流控芯片进行了葡萄糖浓度的电化学流动检测.结果表明,葡萄糖溶液的浓度与响应电流具有较高的线性关系,可对一定浓度范围内的葡萄糖溶液进行定量检测.基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法具有微喷射精度高、重复性好,制备系统结构简单、成本低廉等优点,可用于生化分析、生物传感器等领域的芯片制备.     

Nanomaterials and Analytical Chemistry

Abstract

Nanomaterials play an important role in the area of sensor technology. In fact the sensitivity and the signal‐to‐noise ratio of many chemical sensors are significantly improved using nanomaterials. They have allowed the introduction of many strategies in sensors and biosensor technology. Recently, catalytic nanomotors were used for drug delivery, showing an oriented motion into the cells when they are assembled using magnetic nanowires. In this review, detailed bibliographic references are presented concerning the assembling of nanomaterial‐based sensors, and a brief discussion about the potential health risk of nanoparticles will be also presented.     

The study of nafion/xanthine oxidase/au colloid chemically modified biosensor and its application in the determination of hypoxanthine in myocardial cells in vivo

A novel hypoxanthine (Hx) microsensor was constructed. In this work, Nafion xanthine oxidase (XOD) and Au colloid were immobilized onto the surface of a Pt microelectrode. The enzyme biosensor displayed a quick and sensitive response to Hx. Under physiological conditions, a low detection limit, with high selectivity and sensitivity for Hx determination were obtained. The oxidation current [investigated using current-time (I-t) plots] was linear with Hx concentration ranging from 2.0 x 10(-7) to 2.0 x 10(-5) mol L(-1) with a calculated detection limit of 1.0 x 10(-7) mol L(-1) (S/N of 3). The biosensor should be promising for in vivo measurement of Hx without interferences and fouling. The change of Hx concentration in cardiac myocytes stimulated by L-arginine (L-Arg) and acetylcholine (Ach) was also studied.     

Microsample preparation by dielectrophoresis: isolation of malaria

An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.     

Integration of field effect transistor-based biosensors with a digital microfluidic device for a lab-on-a-chip application

A new platform for lab-on-a-chip system is suggested that utilizes a biosensor array embedded in a digital microfluidic device. With field effect transistor (FET)-based biosensors embedded in the middle of droplet-driving electrodes, the proposed digital microfluidic device can electrically detect avian influenza antibody (anti-AI) in real time by tracing the drain current of the FET-based biosensor without a labeling process. Digitized transport of a target droplet enclosing anti-AI from an inlet to the embedded sensor is enabled by the actuation of electrowetting-on-dielectrics (EWOD). A reduction of the drain current is observed when the target droplet is merged with a pre-existing droplet on the embedded sensor. This reduction of the drain current is attributed to the specific binding of the antigen and the antibody of the AI. The proposed hybrid device consisting of the FET-based sensor and an EWOD device, built on a coplanar substrate by monolithic integration, is fully compatible with current fabrication technology for control and read-out circuitry. Such a completely electrical manner of inducing the transport of bio-molecules, the detection of bio-molecules, the recording of signals, signal processing, and the data transmission process does not require a pump, a fluidic channel, or a bulky transducer. Thus, the proposed platform can contribute to the construction of an all-in-one chip.     

Laser-patterned stem-cell bridges in a cardiac muscle model for on-chip electrical conductivity analyses

Following myocardial infarction there is an irreversible loss of cardiomyocytes that results in the alteration of electrical propagation in the heart. Restoration of functional electrical properties of the damaged heart muscle is essential to recover from the infarction. While there are a few reports that demonstrate that fibroblasts can form junctions that transmit electrical signals, a potential alternative using the injection of stem cells has emerged as a promising cellular therapy; however, stem-cell electrical conductivity within the cardiac muscle fiber is unknown. In this study, an in vitro cardiac muscle model was established on an MEA-based biochip with multiple cardiomyocytes that mimic cardiac tissue structure. Using a laser beam, stem cells were inserted adjacent to each muscle fiber (cell bridge model) and allowed to form cell-cell contact as determined by the formation of gap junctions. The electrical conductivity of stem cells was assessed and compared with the electrical conductivities of cardiomyocytes and fibroblasts. Results showed that stem cell-myocyte contacts exhibited higher and more stable conduction velocities than myocyte-fibroblast contacts, which indicated that stem cells have higher electrical compatibility with native cardiac muscle fibers than cardiac fibroblasts.     

A Review of Biosensor and Industrial Applications of pH-ISFETs and an Evaluation of Honeywell’s “DuraFET”

Biosensors were viewed by earlier workers as possibly lucrative applications for ion selective field effect transistor (ISFET) technology. Such products have yet to find widespread use, however, for a number of reasons that will be reviewed in this article. Some have to do with the inherent nature of biosensor applications per se. For example, biocompatibility is an issue that must be faced regardless of the mechanism of transduction. Others have to do with the inherent nature of the sensor itself. Encapsulation of electronically sensitive parts can prove to be so problematical that lengthy timelines and costly overruns compromisecommercial promise. Intermediate applications, which will be referred to here as “industrial” appear to be commercially viable, however, and can be used to provide profits that will drive further development in the biosensor area. Such applications will also be reviewed. Finally, a specific application will be described in detail (exclusive of proprietary concerns), to illustrate the remarkable ruggedness, stability, and accuracy of a particular commercially available pH-ISFET – Honeywell’s “DuraFET”– a truly useful ion selective field effect transistor.     

Model of a confined spherical cell in uniform and heterogeneous applied electric fields

Cells exposed to electric fields are often confined to a small volume within a solid tissue or within or near a device. Here we report on an approach to describing the frequency and time domain electrical responses of a spatially confined spherical cell by using a transport lattice system model. Two cases are considered: (1) a uniform applied field created by parallel plane electrodes, and (2) a heterogeneous applied field created by a planar electrode and a sharp microelectrode. Here fixed conductivities and dielectric permittivities of the extra- and intracellular media and of the membrane are used to create local transport models that are interconnected to create the system model. Consistent with traditional analytical solutions for spherical cells in an electrolyte of infinite extent, in the frequency domain the field amplification, G(m) (f) is large at low frequencies, f<1 MHz. G(m) (f) gradually decreases above 1 MHz and reaches a lower plateau at about 300 MHz, with the cell becoming almost "electrically invisible". In the time domain the application of a field pulse can result in altered localized transmembrane voltage changes due to a single microelectrode. The transport lattice approach provides modular, multiscale modeling capability that here ranges from cell membranes (5 nm scale) to the cell confinement volume ( approximately 40 microm scale).     

Reagentless biosensor for isocitrate using one step modified Pt-Ir microelectrode

The Pt-Ir microelectrode modified through one step electropolymerization is proposed for the isocitrate amperometric biosensor construction. The enzyme (isocitrate dehydrogenase-ICDH), coenzyme (NADP(+)) and mediator (Meldola's Blue) were immobilized onto the microelectrode surface in one step from a PIPES buffer solution containing pyrrole. The optimized experimental conditions were 25 cycles of cyclic voltammetric in a solution containing 3.58 10(-5) mol l(-1) of mediator, 3.51 10(-4) mol l(-1) of coenzyme and 2.68 U ml(-1) of enzyme. In contrast to the biosensor for isocitrate reported in literature, just one enzyme was immobilized and no coenzyme addition in the solution of analysis was necessary. Catalytic currents were proportional to the isocitrate concentration between 7.7 10(-6) and 1.04 10(-4) mol l(-1), showing good repeatability. The detection limit of the proposed biosensor was 3.50 10(-6) mol l(-1), the response time was lower than 20 s, the lifetime was about 30 determinations and no significant interference of sugars and citric acid was verified. Orange juice samples were analysed by both methodology biosensor and spectrophotometric commercial kit, and the obtained results presented a good correlation. The data demonstrated that the developed biosensor is suitable for isocitrate determination in orange juice without matrix interferences.