CMOS MULTI-MODAL INTEGRATED SYSTEMS FOR FUTURE BIOELECTRONICS AND BIOSENSORS

Cells are the basic structural biological units of all known living organisms. They are highly sophisticated system with thousands of molecules operating in hundreds of pathways to maintain their proper functions, phenotypes, and physiological behaviors. With this scale of complexity, cells often exhibit multi-physiological properties as their cellular fingerprints from external stimulations. In order to further advance the frontiers in bioscience and biotechnologies such as stem cell manufacturing, synthetic biology, and regenerative medicine, it is required to comprehend complex cell physiology of living cells. Therefore, a comprehensive set of technologies is needed to harvest quantitative biological data from given cell samples. Such demands have stimulated extensive research on new bioelectronics and biosensors to characterize their functional information by converting their biological activities to electrical signals. As a result, various bioelectronics and biosensors are reported and employed in many in vivo and in vitro applications. Since sensing electrodes of the devices are physically in touch with biological/chemical samples and record their signals, long-term biocompatibility and chemical/mechanical stability is of paramount importance in numerous biological applications. Furthermore, the devices should achieve high sensitivity/resolution/linearity, large field-of-view (FoV), multi-modal sensing, and real-time monitoring, while maintaining small feature size of devices to use small volume of biological/chemical samples and reduce cost. As a result, My Ph.D research aims to study interfacial electrochemical impedance spectroscopy (EIS) of electrodes with different combination of materials/sizes and to design novel multi-modal sensing/actuation array architectures with CMOS compatible in-house post-processing to address the design challenges of the bioelectronics and biosensors.Ph.D

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

In electrophysiology, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate. In parallel, studies exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. Delivery and recording have independently led to great advances in neurobiology, however, their integration on a single chip would give complete insights into pathologies development and fundamental advancements in drug screening methods. In this work, we demonstrate how a microfluidic-MEA technology may be used to record both spontaneous and chemically induced activity in vitro. We propose a device that can deliver molecules to only a few chosen cells and detecting the response in cellular activity at multiple sites simultaneously. In addition, will be discussed how the adoption of nanoporous metamaterial in place of nanostructures might lower costs and speed up production. Furthermore, this same material, will be identified for the first time in this work as photoelectrical modulating material for eliciting electrogenic cells firing activity. Specifically, by converting NIR laser pulses into stimulatory currents, plasmonic metamaterials may be employed to induce action potentials. This method enables remote access to optical pacing with precise spatiotemporal control, allowing to be used as a valid alternative of the traditional genetic-based optical stimulation techniques. Therefore, in addition to pharmaceutical applications, these final characteristics may pave the way for a new generation of minimally invasive, cellular type-independent all-optical plasmonic pacemakers and muscle actuators

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

Biological studies on in vitro cell cultures are of fundamental importance for investigating cell response to external stimuli, such drugs for specific treatments, or for studying communication between cells. In the electrophysiology field, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. Thus, their improvement is a central topic nowadays in neuroscience and cardiology [1]. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate[2], allowing for monitoring action potentials from many cells simultaneously. In fact, nanoscale biomaterials were able to overcome the limitations of previous technologies, paving the way to the development of platforms for interfacing the electrogenic cells at unprecedented spatiotemporal scales. These devices, together with microfluidics, are starting to be used for drug screening and pharmaceutical drug development since they represent a powerful tool for monitoring cell response when cultures are stimulated by target compounds. Many pharmaceutical agents, however, including various large molecules (enzymes, proteins, antibodies) and even drug-loaded pharmaceutical nanocarriers, need to be delivered intracellularly to exercise their therapeutic action inside the cytoplasm[3]. Nanoscale electrodes offer individual cell access and non-destructive poration of the cellular membrane enabling high capability in the delivery of biomolecules. Among all the techniques, electroporation have proven encouraging potential as alternative to the carrier mediated methods for molecular delivery into cultured cells[4]. In this regard, different groups [5][6][7] exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. These efforts provided powerful tools for advancing applications in therapeutics, diagnostics, and drug discovery, in order to reach an efficient and localized delivery on a chip. Despite these new tactics, there is still a critical need for the development of a functional approach that combines recording capabilities of nanostructured biosensors with intracellular delivery. The device should provide for tight contact between cells and electrode so as to enable highly localized delivery and optimal recording of action potentials in order to attain a high degree of prediction for the disease modeling and drug discovery. This \u201con-chip\u201d approach will help to gain deeper insight in several bio-related studies and analyses, providing a comprehensive knowledge of the entire cellular dynamics when selectively stimulated by the desired bio-molecules. In the first part of this dissertation, a solution will be proposed in order to fill this gap and respond to this need in the biology field. In the first chapter, I will describe briefly the principles of action potentials and how neurons and cardiomyocyte are composed, together with the development of electrophysiology and the advent of multielectrode arrays. In the second chapter, more details about fabrication and cell-electrode system modelling will be explained. In the same chapter, I will explore the development of multielectrode arrays up to the present days, along with the advent of nanotechnologies and the related techniques for improving the previous platforms. The different cell poration techniques will be described in order to reach the best recording capabilities without damaging cells. Electroporation, optoporation and spontaneous poration will be presented and the chosen technique for our application (electroporation) will be reviewed more in detail. In the third chapter, different methodologies for intracellular delivery will be explained, focusing also on the electroporation technique. A small paragraph about the integration of these techniques on chip will be inserted to illustrate the state of the art of these devices. The fourth chapter will explicate in details the Microfluidic multielectrode array idea, the approach used in order to fabricate this novel platform from scratch, the experiments carried out to verify its capabilities and the associated results. In the last paragraph, I will discuss how the proposed platform could became suitable for the day to day uses in research activity by employing nanoporous materials. In fact, big efforts are carried out in order to find appropriate metamaterials as substitutes of the 3D counterparts so as to decrease the cost of device manufacturing that makes them unfitting with research activity. As a novel electrode material, nanoporous metals possess unique properties, such as a low fabrication cost, high plasmonic enhancement and large surface-volume ratio[8]. Nanoporous gold behaves like a metamaterial whose effective dielectric response can be tuned accordingly to the wanted use. These properties make the material suitable for multiple biosensing application, from a high-performance and reliable SERS (surface enhanced raman scattering) substrate [9] to an electrode in CMOS MEAs capable of intracellular recordings[10]. All these properties were explored in the last years, but it could be interesting to further study if the characteristics of this material could make it a good photoelectrical modulating material for eliciting electrogenic cells firing activity. In this way, this technology could be in principle easily implemented on commercial CMOS devices, consenting stimulation and recording at single cell level with high-resolution sensors, opening the way to new methodologies for studying electrogenic cells and tissues. Electrical stimulation of excitable cells is the basis for many implantable devices in cardiac treatment and in neurological studies for treating debilitating neurological syndromes. In order to make the technique less invasive, optical stimulation was widely investigated [11]. The non-genetic photostimulation is starting to make its way in the field since it allows to avoid changing the biological framework by using transient thermal or electrochemical outputs from synthetic materials attached to the target cells[12]. If stimulated with impinging light these materials could inject free charges into the solution resulting in an ionic current at the interface able to eliciting of neurons[13] or cardiomyocyte action potentials. Plasmonic porous materials have all the suitable properties to be considered as an appealing tools for charge injection and consequently for stimulation of electrically active cells [14]. Thus, the second part of this dissertation will exploit the capabilities of these plasmonic metamaterials, placing particular emphasis on the possibility of photoelectrochemical modulation. In particular, in the fifth and last chapter I will describe all the properties and application of the porous material and the mechanism of photoemission. In the experimental paragraphs, the free charge photoemission properties of porous gold will be explored together with plasmonic non-genetic photostimulation of the cardiac cells on commercial CMOS MEAs

The EcoChip : a wireless multi-sensor platform for comprehensive environmental monitoring

This paper presents the EcoChip, a new system based on state-of-the-art electro-chemical impedance (EIS) technologies allowing the growth of single strain organisms isolated from northern habitats. This portable system is a complete and autonomous wireless platform designed to monitor and cultivate microorganisms directly sampled from their natural environment, particularly from harsh northern environments. Using 96-well plates, the EcoChip can be used in the field for realtime monitoring of bacterial growth. Manufactured with highquality electronic components, this new EIS monitoring system is designed to function at a low excitation voltage signal to avoid damaging the cultured cells. The high-precision calibration network leads to high-precision results, even in the most limiting contexts. Luminosity, humidity and temperature can also be monitored with the addition of appropriate sensors. Access to robust data storage systems and power supplies is an obvious limitation for northern research. That is why the EcoChip is equipped with a flash memory that can store data over long periods of time. To resolve the power issue, a low-power microcontroller and a power management unit control and supply all electronic building blocks. Data stored in the EcoChip’s flash memory can be transmitted through a transceiver whenever a receiver is located within the functional transmission range. In this paper, we present the measured performance of the system, along with results from laboratory tests in-vitro and from two field tests. The EcoChip has been utilized to collect bio-environemental data in the field from the northern soils and ecosystems of Kuujjuarapik and Puvirnituq, during two expeditions, in 2017 and 2018, respectively. We show that the EcoChip can effectively carry out EIS analyses over an excitation frequency ranging from 750 Hz to 10 kHz with an accuracy of 2.35%. The overall power consumption of the system was 140.4 mW in normal operating mode and 81 µW in sleep mode. The proper development of the isolated bacteria was confirmed through DNA sequencing, indicating that bacteria thrive in the EcoChip’s culture wells while the growing conditions are successfully gathered and stored

Time Stamp – A Novel Time-to-Digital Demodulation Method for Bioimpedance Implant Applications

Bioimpedance analysis is a noninvasive and inexpensive technology used to investigate the electrical properties of biological tissues. The analysis requires demodulation to extract the real and imaginary parts of the impedance. Conventional systems use complex architectures such as I-Q demodulation. In this paper, a very simple alternative time-to-digital demodulation method or ‘time stamp’ is proposed. It employs only three comparators to identify or stamp in the time domain, the crossing points of the excitation signal, and the measured signal. In a CMOS proof of concept design, the accuracy of impedance magnitude and phase is 97.06% and 98.81% respectively over a bandwidth of 10 kHz to 500 kHz. The effect of fractional-N synthesis is analysed for the counter-based zero crossing phase detector obtaining a finer phase resolution (0.51˚ at 500 kHz) using a counter clock frequency ( fclk = 12.5 MHz). Because of its circuit simplicity and ease of transmitting the time stamps, the method is very suited to implantable devices requiring low area and power consumption

Multimodal integrated sensor platform for rapid biomarker detection

Precision metabolomics and quantification for cost-effective, rapid diagnosis of disease are key goals in personalized medicine and point-of-care testing. Presently, patients are subjected to multiple test procedures requiring large laboratory equipment. Microelectronics has already made modern computing and communications possible by integration of complex functions within a single chip. As More than Moore technology increases in importance, integrated circuits for densely patterned sensor chips have grown in significance. Here, we present a versatile single CMOS chip forming a platform to address personalized needs through on-chip multimodal optical and electrochemical detection that will reduce the number of tests that patients must take. The chip integrates interleaved sensing subsystems for quadruple-mode colorimetric, chemiluminescent, surface plasmon resonance and hydrogen ion measurements. These subsystems include a photodiode array and a single photon avalanche diode array, with some elements functionalized to introduce a surface plasmon resonance mode. The chip also includes an array of ion sensitive field effect transistors. The sensor arrays are distributed uniformly over an active area on the chip surface in a scalable and modular design. Bio-functionalization of the physical sensors yields a highly selective simultaneous multiple-assay platform in a disposable format. We demonstrate its versatile capabilities through quantified bioassays performed on-chip for glucose, cholesterol, urea and urate, each within their naturally occurring physiological range

A 4.8-μVrms-Noise CMOS-Microelectrode Array With Density-Scalable Active Readout Pixels via Disaggregated Differential Amplifier Implementation

We demonstrate a 4.8-μVrms noise microelectrode array (MEA) based on the complementary-metal-oxide-semiconductor active-pixel-sensors readout technique with disaggregated differential amplifier implementation. The circuit elements of the differential amplifier are divided into a readout pixel, a reference pixel, and a column circuit. This disaggregation contributes to the small area of the readout pixel, which is less than 81 μm2. We observed neuron signals around 100 μV with 432 electrodes in a fabricated prototype chip. The implementation has technological feasibility of up to 12-μm-pitch electrode density and 6,912 readout channels for high-spatial resolution mapping of neuron network activity

Development of Facile Microfabrication Technologies for the Fabrication and Characterization of Multimodal Impedimetric, Plasmonic, and Electrophysiological Biosensors

The objective of this dissertation was to develop novel methods of patterning inorganic and organic materials, develop biocompatibility evaluations, and subsequently apply these methods toward developing biosensors and lab-on-a-chip devices, such as Interdigitated Electrodes (IDEs) and Microelectrode Arrays (MEAs) on non-traditional (such as nanostructured and plasmonic) polymer substrates or deploy these methods to enhance precision cellular placement on traditional (glass) MEA substrates. It was hypothesized that a combination of such facile microfabrication techniques and patterning technologies on traditional and non-traditional substrates would increase the sensitivity and selectivity of such sensor platforms by several orders of magnitude, and potentially introduce new modalities for cell-based biosensing. In order to demonstrate the biological functionality of these new IDEs and MEAs, a variety of cell cultures were used (cardiac, stem cell, and endothelial cells) to study the growth, proliferation, modes of increasing sensitivity and response to various compounds in vitro (outside the body)

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