Digital CMOS ISFET architectures and algorithmic methods for point-of-care diagnostics

Over the past decade, the surge of infectious diseases outbreaks across the globe is redefining how healthcare is provided and delivered to patients, with a clear trend towards distributed diagnosis at the Point-of-Care (PoC). In this context, Ion-Sensitive Field Effect Transistors (ISFETs) fabricated on standard CMOS technology have emerged as a promising solution to achieve a precise, deliverable and inexpensive platform that could be deployed worldwide to provide a rapid diagnosis of infectious diseases. This thesis presents advancements for the future of ISFET-based PoC diagnostic platforms, proposing and implementing a set of hardware and software methodologies to overcome its main challenges and enhance its sensing capabilities. The first part of this thesis focuses on novel hardware architectures that enable direct integration with computational capabilities while providing pixel programmability and adaptability required to overcome pressing challenges on ISFET-based PoC platforms. This section explores oscillator-based ISFET architectures, a set of sensing front-ends that encodes the chemical information on the duty cycle of a PWM signal. Two initial architectures are proposed and fabricated in AMS 0.35um, confirming multiple degrees of programmability and potential for multi-sensing. One of these architectures is optimised to create a dual-sensing pixel capable of sensing both temperature and chemical information on the same spatial point while modulating this information simultaneously on a single waveform. This dual-sensing capability, verified in silico using TSMC 0.18um process, is vital for DNA-based diagnosis where protocols such as LAMP or PCR require precise thermal control. The COVID-19 pandemic highlighted the need for a deliverable diagnosis that perform nucleic acid amplification tests at the PoC, requiring minimal footprint by integrating sensing and computational capabilities. In response to this challenge, a paradigm shift is proposed, advocating for integrating all elements of the portable diagnostic platform under a single piece of silicon, realising a ``Diagnosis-on-a-Chip". This approach is enabled by a novel Digital ISFET Pixel that integrates both ADC and memory with sensing elements on each pixel, enhancing its parallelism. Furthermore, this architecture removes the need for external instrumentation or memories and facilitates its integration with computational capabilities on-chip, such as the proposed ARM Cortex M3 system. These computational capabilities need to be complemented with software methods that enable sensing enhancement and new applications using ISFET arrays. The second part of this thesis is devoted to these methods. Leveraging the programmability capabilities available on oscillator-based architectures, various digital signal processing algorithms are implemented to overcome the most urgent ISFET non-idealities, such as trapped charge, drift and chemical noise. These methods enable fast trapped charge cancellation and enhanced dynamic range through real-time drift compensation, achieving over 36 hours of continuous monitoring without pixel saturation. Furthermore, the recent development of data-driven models and software methods open a wide range of opportunities for ISFET sensing and beyond. In the last section of this thesis, two examples of these opportunities are explored: the optimisation of image compression algorithms on chemical images generated by an ultra-high frame-rate ISFET array; and a proposed paradigm shift on surface Electromyography (sEMG) signals, moving from data-harvesting to information-focused sensing. These examples represent an initial step forward on a journey towards a new generation of miniaturised, precise and efficient sensors for PoC diagnostics.Open Acces

A dual-sensing thermo-chemical ISFET array for DNA-based diagnostics.

This paper presents a 32x32 ISFET array with in-pixel dual-sensing and programmability targeted for on-chip DNA amplification detection. The pixel architecture provides thermal and chemical sensing by encoding temperature and ion activity in a single output PWM, modulating its frequency and its duty cycle respectively. Each pixel is composed of an ISFET-based differential linear OTA and a 2-stage sawtooth oscillator. The operating point and characteristic response of the pixel can be programmed, enabling trapped charge compensation and enhancing the versatility and adaptability of the architecture. Fabricated in 0.18 μm standard CMOS process, the system demonstrates a quadratic thermal response and a highly linear pH sensitivity, with a trapped charge compensation scheme able to calibrate 99.5% of the pixels in the target range, achieving a homogeneous response across the array. Furthermore, the sensing scheme is robust against process variations and can operate under various supply conditions. Finally, the architecture suitability for on-chip DNA amplification detection is proven by performing Loop-mediated Isothermal Amplification (LAMP) of phage lambda DNA, obtaining a time-to-positive of 7.71 minutes with results comparable to commercial qPCR instruments. This architecture represents the first in-pixel dual thermo-chemical sensing in ISFET arrays for Lab-on-a-Chip diagnostics

Wearable, low-power CMOS ISFETs and compensation circuits for on-body sweat analysis

Complementary metal-oxide-semiconductor (CMOS) technology has been a key driver behind the trend of reduced power consumption and increased integration of electronics in consumer devices and sensors. In the late 1990s, the integration of ion-sensitive field-effect transistors (ISFETs) into unmodified CMOS helped to create advancements in lab-on-chip technology through highly parallelised and low-cost designs. Using CMOS techniques to reduce power and size in chemical sensing applications has already aided the realisation of portable, battery-powered analysis platforms, however the possibility of integrating these sensors into wearable devices has until recently remained unexplored. This thesis investigates the use of CMOS ISFETs as wearable electrochemical sensors, specifically for on-body sweat analysis. The investigation begins by evaluating the ISFET sensor for wearable applications, identifying the key advantages and challenges that arise in this pursuit. A key requirement for wearable devices is a low power consumption, to enable a suitable operational life and small form factor. From this perspective, ISFETs are investigated for low power operation, to determine the limitations when trying to push down the consumption of individual sensors. Batteryless ISFET operation is explored through the design and implementation of a 0.35 \si{\micro\metre} CMOS ISFET sensing array, operating in weak-inversion and consuming 6 \si{\micro\watt}. Using this application-specific integrated circuit (ASIC), the first ISFET array powered by body heat is demonstrated and the feasibility of using near-field communication (NFC) for wireless powering and data transfer is shown. The thesis also presents circuits and systems for combatting three key non-ideal effects experienced by CMOS ISFETs, namely temperature variation, threshold voltage offset and drift. An improvement in temperature sensitivity by a factor of three compared to an uncompensated design is shown through measured results, while adding less than 70 \si{\nano\watt} to the design. A method of automatically biasing the sensors is presented and an approach to using spatial separation of sensors in arrays in applications with flowing fluids is proposed for distinguishing between signal and sensor drift. A wearable device using the ISFET-based system is designed and tested with both artificial and natural sweat, identifying the remaining challenges that exist with both the sensors themselves and accompanying components such as microfluidics and reference electrode. A new ASIC is designed based on the discoveries of this work and aimed at detecting multiple analytes on a single chip. %Removed In the latter half of the thesis, Finally, the future directions of wearable electrochemical sensors is discussed with a look towards embedded machine learning to aid the interpretation of complex fluid with time-domain sensor arrays. The contributions of this thesis aim to form a foundation for the use of ISFETs in wearable devices to enable non-invasive physiological monitoring.Open Acces

Quantitative detection of dengue serotypes using a smartphone-connected handheld lab-on-chip platform

Dengue is one of the most prevalent infectious diseases in the world. Rapid, accurate and scalable diagnostics are key to patient management and epidemiological surveillance of the dengue virus (DENV), however current technologies do not match required clinical sensitivity and specificity or rely on large laboratory equipment. In this work, we report the translation of our smartphone-connected handheld Lab-on-Chip (LoC) platform for the quantitative detection of two dengue serotypes. At its core, the approach relies on the combination of Complementary Metal-Oxide-Semiconductor (CMOS) microchip technology to integrate an array of 78 × 56 potentiometric sensors, and a label-free reverse-transcriptase loop mediated isothermal amplification (RT-LAMP) assay. The platform communicates to a smartphone app which synchronises results in real time with a secure cloud server hosted by Amazon Web Services (AWS) for epidemiological surveillance. The assay on our LoC platform (RT-eLAMP) was shown to match performance on a gold-standard fluorescence-based real-time instrument (RT-qLAMP) with synthetic DENV-1 and DENV-2 RNA and extracted RNA from 9 DENV-2 clinical isolates, achieving quantitative detection in under 15 min. To validate the portability of the platform and the geo-tagging capabilities, we led our study in the laboratories at Imperial College London, UK, and Kaohsiung Medical Hospital, Taiwan. This approach carries high potential for application in low resource settings at the point of care (PoC)

Handheld ISFET Lab-on-Chip detection of TMPRSS2-ERG and AR mRNA for prostate cancer prognostics

Ion-sensitive field-effect transistors (ISFETs) in combination with unmodified complementary metal oxide semiconductors present a point-of-care platform for clinical diagnostics and prognostics. This work illustrates the sensitive and specific detection of two circulating mRNA markers for prostate cancer, the androgen receptor and the TMPRSS2-ERG fusion using a target-specific loop-mediated isothermal amplification method. TMPRSS2-ERG and androgen receptor RNA were detected down to 3x10 1 and 5x10 1 copies respectively in under 30 minutes. Administration of these assays onto the ISFET Lab-on-chip device was successful and the specificity of each marker was corroborated with mRNA extracted from prostate cancer cell lines

Rapid detection of mobilized colistin resistance using a nucleic acid based lab-on-a-chip diagnostic system

The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of circulating cell-free DNA fragments in the patients’ blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy. Next generation sequencing platforms and digital droplet PCR can be used to profile circulating tumour DNA from liquid biopsies; however, they are expensive and time consuming for clinical use. Here, we report a novel strategy with proof-of-concept data that supports the usage of loop-mediated isothermal amplification (LAMP) to detect PIK3CA c.3140 A > G (H1047R), a prevalent BC missense mutation that is attributed to BC tumour growth. Allele-specific primers were designed and optimized to detect the p.H1047R variant following the USS-sbLAMP method. The assay was developed with synthetic DNA templates and validated with DNA from two breast cancer cell-lines and two patient tumour tissue samples through a qPCR instrument and finally piloted on an ISFET enabled microchip. This work sets a foundation for BC mutational profiling on a Lab-on-Chip device, to help the early detection of patient relapse and to monitor efficacy of systemic therapies for personalised cancer patient management

A Label Free CMOS-Based Smart Petri Dish for Cellular Analysis

RÉSUMÉ Le dépistage de culture cellulaire à haut débit est le principal défi pour une variété d’applications des sciences de la vie, y compris la découverte de nouveaux médicaments et le suivi de la cytotoxicité. L’analyse classique de culture cellulaire est généralement réalisée à l’aide de techniques microscopiques non-intégrées avec le système de culture cellulaire. Celles-ci sont laborieuses spécialement dans le cas des données recueillies en temps réel ou à des fins de surveillance continue. Récemment, les micro-réseaux cellulaires in-vitro ont prouvé de nombreux avantages dans le domaine de surveillance des cellules en réduisant les coûts, le temps et la nécessité d’études sur des modèles animaux. Les microtechniques, y compris la microélectronique et la microfluidique,ont été récemment utilisé dans la biotechnologie pour la miniaturisation des systèmes biologiques et analytiques. Malgré les nombreux efforts consacrés au développement de dispositifs microfluidiques basés sur les techniques de microscopie optique, le développement de capteurs intégrés couplés à des micropuits pour le suivi des paramètres cellulaires tel que la viabilité, le taux de croissance et cytotoxicité a été limité. Parmi les différentes méthodes de détection disponibles, les techniques capacitives offrent une plateforme de faible complexité. Celles-ci ont été considérablement utilisées afin d’étudier l’interaction cellule-surface. Ce type d’interaction est le plus considéré dans la majorité des études biologiques. L’objectif de cette thèse est de trouver des nouvelles approches pour le suivi de la croissance cellulaire et la surveillance de la cytotoxicité à l’aide d’un réseau de capteurs capacitifs entièrement intégré. Une plateforme hybride combinant un circuit microélectronique et une structure microfluidique est proposée pour des applications de détection de cellules et de découverte de nouveaux médicaments. Les techniques biologiques et chimiques nécessaires au fonctionnement de cette plateforme sont aussi proposées. La technologie submicroniques Standard complementary metal-oxide-Semiconductor (CMOS) (TSMC 0.35 μm) est utilisée pour la conception du circuit microélectronique de cette plateforme. En outre, les électrodes sont fabriquées selon le processus CMOS standard sans la nécessité d’étapes de post-traitement supplémentaires. Ceci rend la plateforme proposée unique par rapport aux plateformes de dépistage de culture cellulaire à haut débit existantes. Plusieurs défis ont été identifiés durant le développement de cette plateforme comme la sensibilité, la bio-compatibilité et la stabilité et les solutions correspondantes sont fournies.----------ABSTRACT High throughput cell culture screening is a key challenge for a variety of life science applications, including drug discovery and cytotoxicity monitoring. Conventional cell culture analysis is widely performed using microscopic techniques that are not integrated into the target cell culture system. Additionally, these techniques are too laborious in particular to be used for real-time and continuous monitoring purposes. Recently, it has been proved that invitro cell microarrays offer great advantages for cell monitoring applications by reducing cost, time, and the need for animal model studies. Microtechnologies, including microelectronics and microfluidics, have been recently used in biotechnology for miniaturization of biological and analytical systems. Despite many efforts in developing microfluidic devices using optical microscopy techniques, less attention have been paid on developing fully integrated sensors for monitoring cell parameters such as viability, growth rate, and cytotoxicity. Among various available sensing methods, capacitive techniques offer low complexity platforms. This technique has significantly attracted attentions for the study of cell-surface interaction which is widely considered in biological studies. This thesis focuses on new approaches for cell growth and cytotoxicity monitoring using a fully integrated capacitive sensor array. A hybrid platform combining microelectronic circuitry and microfluidic structure is proposed along with other required biological and chemical techniques for single cell detection and drug discovery applications. Standard submicron complementary metal–oxide–semiconductor (CMOS) technology (TSMC 0.35 μm) is used to develop the microelectronic part of this platform. Also, the sensing electrodes are fabricated in standard CMOS process without the need for any additional post processing step, which makes the proposed platform unique compared to other state of the art high throughput cell assays. Several challenges in implementing this platform such as sensitivity, bio-compatibility, and stability are discussed and corresponding solutions are provided. Specifically, a new surface functionalization method based on polyelectrolyte multilayers deposition is proposed to enhance cell-electrode adherence and to increase sensing electrodes’ life time. In addition, a novel technique for microwell fabrication and its integration with the CMOS chip is proposed to allow parallel screening of cells. With the potential to perform inexpensive, fast, and real-time cell analyses, the proposed platform opens up the possibility to transform from passive traditional cell assays to a smart on-line monitoring system

Graphene inspired sensing devices

Graphene’s exciting characteristics such as high mechanical strength, tuneable electrical prop- erties, high thermal conductivity, elasticity, large surface-to-volume ratio, make it unique and attractive for a plethora of applications including gas and liquid sensing. Adsorption, the phys- ical bonding of molecules on solid surfaces, has huge impact on the electronic properties of graphene. We use this to develop gas sensing devices with faster response time by suspending graphene over large area (cm^2) on silicon nanowire arrays (SiNWAs). These are fabricated by two-step metal-assisted chemical etching (MACE) and using a home-developed polymer-assisted graphene transfer (PAGT) process. The advantage of suspending graphene is the removal of diffusion-limited access to the adsorption sites at the interface between graphene and its support. By modifying the Langmuir adsorption model and fitting the experimental response curves, we find faster response times for both ammonia and acetone vapours. The use of suspended graphene improved the overall response, based on speed and amplitude of response, by up to 750% on average. This device could find applications in biomedical breath analysis for diseases such lung cancer, asthma, kidney failure and more. Taking advantage of the mechanical strength of graphene and using the developed PAGT process, we transfer it on commercial (CMOS) Ion-Sensitive Field-Effect Transistor (ISFET) arrays. The deposition of graphene on the top sensing layer reduces drift that results from the surface modification during exposure to electrolyte while improving the overall performance by up to about 10^13 % and indicates that the ISFET can operate with metallic sensing membrane and not only with insulating materials as confirmed by depositing Au on the gate surface. Post- processing of the ISFET top surface by reactive ion plasma etching, proved that the physical location of trapped charge lies within the device structure. The process improved its overall performance by about 105 %. The post-processing of the ISFET could be applied for sensor performance in any of its applications including pH sensing for DNA sequencing and glucose monitoring.Open Acces

Electrochemical sensor system architecture using the CMOS-MEMS technology for cytometry applications

This thesis presents the development process of an integrated sensor-system-on-chip for recording the parameters of blood cells. The CMOS based device consists of the two flow-through sensor arrays, stacked one on top of the other. The sensors are able to detect the biological cell in terms of its physical size and the surface charge on a cell’s membrane. The development of the measurement system was divided into several stages these were to design and implement the two sensor arrays complemented with readout circuitry onto a single CMOS chip to create an on-chip membrane with embedded flow-through micro-channels by a CMOS compatible post-processing techniques to encapsulate and hermeti-cally package the device for liquid chemistry experiments, to test and characterise the two sensor arrays together with readout electronics, to develop control and data acquisition software and to detect the biological cells using the complete measurement system. Cy-tometry and haematology fields are closely related to the presented work, hence it is envis-aged that the developed technology enables further integration and miniaturisation of the biomedical instrumentation. The two vertically stacked 4 x 4 flow-through sensor arrays, embedded into an on-chip membrane, were implemented in a single silicon chip device together with a readout circuitry for each of the sensor sets. To develop a CMOS-MEMS device the design and fabrication was carried out using a commercial process design kit (0.35 µm 4-Metal, 2-Poly, CMOS) as well as the foundry service. Thereafter the device was post-processed in-house to develop the on-chip membrane and open the sensing micro-apertures. The two types of sensor were integrated on the silicon dice for multi-parametric characterisation of the analyte. To read the cell membrane charge the ion sensitive field effect transistor (ISFET) was utilised and for cell size (volume) detection an impedance sensor (Coulter counter) was used. Both sensors rely on a flow-through mode of operation, hence the constant flow of the analyte sample could be maintained. The Coulter counter metal electrode was exposed to the solution, while the ISFET floating gate electrode maintained contact with the analyte through a charge sensitive membrane constructed of a dielectric material (silicon dioxide) lining the inside of the micro-pore. The outside size of each of the electrodes was 100 µm x 100 µm and the inside varied from 20 µm x 20 µm to 58 µm x 58 µm. The sense aperture size also varied from 10 µm x 10 µm to 16 µm x 16 µm. The two stacked micro-electrode arrays were layed out on an area of 5002 µm2. The CMOS-MEMS device was fit into a custom printed circuit board (PCB) chip carrier, thereafter insulated and hermetically packaged. Microfluidic ports were attached to the packaged module so that the analyte can be introduced and drained by a flow-through mode of operation. The complete microfluidic system and packaging was assembled and thereafter evaluated for correct operation. Undisturbed flow of the analyte solution is es-sential for the sensor operation. This is related to the fact that the electrochemical response of both sensors depends on the analyte flow through the sense micro-apertures thus any aggregation of the sample within the microfluidic system would cause clogging of the mi-cro-pores. The on-chip electronic circuitry was characterised, and after comparison with the simulated results found to be within an error margin of what enables it for reliable sensor signal readout. The measurement system is automated by software control so that the bias parame-ters can be set precisely, it also helped while error debugging. Analogue signals from the two sensor arrays were acquired, later processed and stored by a data acquisition system. Both control and data capture systems are implemented in a high level programming lan-guage. Furthermore both are integrated and operated in a one window based graphical user interface (GUI). A fully functional measurement system was used as a flow-through cytometer for living cells detection. The measurements results showed that the system is capable of single cell detection and on-the-fly data display