A 16 x 16 CMOS amperometric microelectrode array for simultaneous electrochemical measurements

There is a requirement for an electrochemical sensor technology capable of making multivariate measurements in environmental, healthcare, and manufacturing applications. Here, we present a new device that is highly parallelized with an excellent bandwidth. For the first time, electrochemical cross-talk for a chip-based sensor is defined and characterized. The new CMOS electrochemical sensor chip is capable of simultaneously taking multiple, independent electroanalytical measurements. The chip is structured as an electrochemical cell microarray, comprised of a microelectrode array connected to embedded self-contained potentiostats. Speed and sensitivity are essential in dynamic variable electrochemical systems. Owing to the parallel function of the system, rapid data collection is possible while maintaining an appropriately low-scan rate. By performing multiple, simultaneous cyclic voltammetry scans in each of the electrochemical cells on the chip surface, we are able to show (with a cell-to-cell pitch of 456 μm) that the signal cross-talk is only 12% between nearest neighbors in a ferrocene rich solution. The system opens up the possibility to use multiple independently controlled electrochemical sensors on a single chip for applications in DNA sensing, medical diagnostics, environmental sensing, the food industry, neuronal sensing, and drug discovery

Biosensor system with an integrated CMOS microelectrode array for high spatio-temporal electrochemical imaging, A

2019 Fall.Includes bibliographical references.The ability to view biological events in real time has contributed significantly to research in life sciences. While optical microscopy is important to observe anatomical and morphological changes, it is equally important to capture real-time two-dimensional (2D) chemical activities that drive the bio-sample behaviors. The existing chemical sensing methods (i.e. optical photoluminescence, magnetic resonance, and scanning electrochemical), are well-established and optimized for existing ex vivo or in vitro analyses. However, such methods also present various limitations in resolution, real-time performance, and costs. Electrochemical method has been advantageous to life sciences by supporting studies and discoveries in neurotransmitter signaling and metabolic activities in biological samples. In the meantime, the integration of Microelectrode Array (MEA) and Complementary-Metal-Oxide-Semiconductor (CMOS) technology to the electrochemical method provides biosensing capabilities with high spatial and temporal resolutions. This work discusses three related subtopics in this specific order: improvements to an electrochemical imaging system with 8,192 sensing points for neurotransmitter sensing; comprehensive design processes of an electrochemical imaging system with 16,064 sensing points based on the previous system; and the application of the system for imaging oxygen concentration gradients in metabolizing bovine oocytes. The first attempt of high spatial electrochemical imaging was based on an integrated CMOS microchip with 8,192 configurable Pt surface electrodes, on-chip potentiostat, on-chip control logic, and a microfluidic device designed to support ex vivo tissue experimentation. Using norepinephrine as a target analyte for proof of concept, the system is capable of differentiating concentrations of norepinephrine as low as 8µM and up to 1,024 µM with a linear response and a spatial resolution of 25.5×30.4μm. Electrochemical imaging was performed using murine adrenal tissue as a biological model and successfully showed caffeine-stimulated release of catecholamines from live slices of adrenal tissue with desired spatial and temporal resolutions. This system demonstrates the capability of an electrochemical imaging system capable of capturing changes in chemical gradients in live tissue slices. An enhanced system was designed and implemented in a CMOS microchip based on the previous generation. The enhanced CMOS microchip has an expanded sensing area of 3.6×3.6mm containing 16,064 Pt electrodes and the associated 16,064 integrated read channels. The novel three-electrode electrochemical sensor system designed at 27.5×27.5µm pitch enables spatially dense cellular level chemical gradient imaging. The noise level of the on-chip read channels allow amperometric linear detection of neurotransmitter (norepinephrine) concentrations from 4µM to 512µM with 4.7pA/µM sensitivity (R=0.98). Electrochemical response to dissolved oxygen concentration or oxygen partial pressure (pO2) was also characterized with deoxygenated deionized water containing 10µM to 165 µM pO2 with 8.21pA/µM sensitivity (R=0.89). The enhanced biosensor system also demonstrates selectivity to different target analytes using cyclic voltammetry to simultaneously detect NE and uric acid. In addition, a custom-designed indium tin oxide and Au glass electrode is integrated into the microfluidic support system to enable pH measurement, ensuring viability of bio-samples in ex vivo experiments. Electrochemical images confirm the spatiotemporal performance at four frames per second while maintaining the sensitivity to target analytes. The overall system is controlled and continuously monitored by a custom-designed user interface, which is optimized for real-time high spatiotemporal resolution chemical bioimaging. It is well known that physiological events related to oxygen concentration gradients provide valuable information to determine the state of metabolizing biological cells. Utilizing the CMOS microchip with 16,064 Pt MEA and an improved three-electrode system configuration, the system is capable of imaging low oxygen concentration with limit of detection of 18.3µM, 0.58mg/L, or 13.8mmHg. A modified microfluidic support system allows convenient bio-sample handling and delivery to the MEA surface for sensing. In vitro oxygen imaging experiments were performed using bovine cumulus-oocytes-complexes cells with custom software algorithms to analyze its flux density and oxygen consumption rate. The imaging results are processed and presented as 2D heatmaps, representing the dissolved oxygen concentration in the immediate proximity of the cell. The 2D images and analysis of oxygen consumption provide a unique insight into the spatial and temporal dynamics of cell metabolism

A microscopy technique based on bio-impedance sensors

It is proposed a microscopy for cell culture applications based on impedance sensors. The imagined signals are measured with the Electrical Cell-Substrate Spectroscopy (ECIS) technique, by identifying the cell area. The proposed microscopy allows real-time monitoring inside the incubator, reducing the contamination risk by human manipulation. It requires specific circuits for impedance measurements, a two-dimensional sensor array (pixels), and employing electrical models to decode efficiently the measured signals. Analogue Hardware Description Language (AHDL) circuits for cell-microelectrode enables the use of geometrical and technological data into the system design flow. A study case with 8x8 sensor array is reported, illustrating the evolution and power of the proposed image acquisition.Junta de Andalucía P0-TIC-538

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

Micro- and nano-devices for electrochemical sensing

Electrode miniaturization has profoundly revolutionized the field of electrochemical sensing, opening up unprecedented opportunities for probing biological events with a high spatial and temporal resolution, integrating electrochemical systems with microfluidics, and designing arrays for multiplexed sensing. Several technological issues posed by the desire for downsizing have been addressed so far, leading to micrometric and nanometric sensing systems with different degrees of maturity. However, there is still an endless margin for researchers to improve current strategies and cope with demanding sensing fields, such as lab-on-a-chip devices and multi-array sensors, brain chemistry, and cell monitoring. In this review, we present current trends in the design of micro-/nano-electrochemical sensors and cutting-edge applications reported in the last 10 years. Micro- and nanosensors are divided into four categories depending on the transduction mechanism, e.g., amperometric, impedimetric, potentiometric, and transistor-based, to best guide the reader through the different detection strategies and highlight major advancements as well as still unaddressed demands in electrochemical sensing

Design, development and characterization of nanostructured electrochemical sensors

This is a publication-based thesis which focuses on the study of electrochemical microbiosensors for glucose detection. It investigates applications of a series of microfabricated gold electrodes based on several nanostructures in electrochemical biosensing technologies, embracing three major methodologies: direct electro-catalytic detection, enzymatic detection and dual-enzyme cascade detection. The study is described over five main chapters with a sixth providing a summary of the material presented and perspectives for the future. Chapter 1 provides an introduction to the field of the electrochemical biosensors with a specific focus on the chosen nanostructures and miniaturized systems, as well as a brief history of the biosensor. Chapter 2 presents results published in ACS Applied Nanomaterials, 2019, 2, 9, 5878-5889. It demonstrates the enzyme free detection of glucose via a direct electro-catalytic reaction. The miniaturized band array electrodes with specific width, length and inter-electrode-distance were integrated with homogeneously distributed copper foam nano dendrites. Such foam deposits presented for the first time at the micro scale were achieved using the in-situ hydrogen bubble template method. The resulting very high electroactive surface area of the porous foam deposits was one of the major advantages in terms of achieving superior performance from each micro band foam electrode towards glucose detection. Moreover, both sensors also showed a strong resistance to the poisoning effects of chloride ions and displayed excellent stability over a period of three months.Chapter 3 presents the first of t wo sets of results for the enzymatic detection of glucose, results published in Elsevier Electrochimica Acta, 2019, 293, 307-317. Chapter 4 then presents the second set of results on this topic which is published in and Elsevier Electrochimica Acta, 2019, 298, 97-105. The aim of these two chapters is to discuss the effect of miniaturization on the enzymatic biosensor performance which was studied in the presence of a carbon quantum dot (CQD) and gold nanoparticle nanohybrid system. CQDs, are a new class of carbon-based materials and have been used here for the first time as a matrix component integrated onto microfabricated gold electrode surfaces for enzyme immobilization and further miniaturization. The biosensors developed were studied by electrochemistry to investigate the analytical performance of each device. By scaling down the surface area of the biosensor, a 13-times increase in sensitivity was achieved towards glucose. Moreover both sensors-planar, micro disk array- exhibited excellent reproducibility, reusability and operational stability in terms of the performance of biosensors. Chapter 5 presents results published in RSC Analyst, 2020 (DOI: 10.1039/C9AN01664C). It demonstrates the operation of a dual-enzyme cascade which was constructed onto a micro band array electrode based on glucose oxidase and horseradish peroxidase enzymes. To achieve a very high surface area, a porous gold-foam was electrodeposited onto surface and then a second electrodeposition layer of chitosan and multi walled carbon nanotube nano-bio-composite. The micro band cascade scheme developed exhibited the highest sensitivity towards glucose detection in comparison to other systems reported in the literature. Chapter 6 provides an insight into the field of electrochemical biosensing with the support of the achievements presented in this thesis. Thus, by taking advantage of the available system, this chapter discusses the possible future applications of the electrochemical biosensors. The thesis then ends with section 7 which presents some Appendices

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 retin

Electrochemical Detection of Neurotransmitters

Neurotransmitters are important chemical messengers in the nervous system that play a crucial role in physiological and physical health. Abnormal levels of neurotransmitters have been correlated with physical, psychotic, and neurodegenerative diseases such as Alzheimer\u27s, Parkinson\u27s, dementia, addiction, depression, and schizophrenia. Although multiple neurotechnological approaches have been reported in the literature, the detection and monitoring of neurotransmitters in the brain remains a challenge and continues to garner significant attention. Neurotechnology that provides high-throughput, as well as fast and specific quantification of target analytes in the brain, without negatively impacting the implanted region is highly desired for the monitoring of the complex intercommunication of neurotransmitters. Therefore, it is crucial to develop clinical assessment techniques that are sensitive and reliable to monitor and modulate these chemical messengers and screen diseases. This review focuses on summarizing the current electrochemical measurement techniques that are capable of sensing neurotransmitters with high temporal resolution in real time. Advanced neurotransmitter sensing platforms that integrate nanomaterials and biorecognition elements are explored