DNA Logic-A novel approach to semiconductor based genetics

In the coming years, genetic test results will be increasingly used as indicators that influence medical decision-making. With chronic disease on the rise and the continuing global spread of infectious disease, novel instruments able to detect relevant mutations in a point-of-care setting are being developed to facilitate this increased demand in personalized health care. However, diagnosis for such demand often requires laboratory facilities and skilled personnel, meaning that diagnostic tests are restricted by time and access. This thesis presents a novel configuration for Ion sensitive Field Effect Transistors (ISFETs) to be used as a threshold detector during nucleic acid base pairs match. ISFET-based inverters are used as reaction threshold detectors to convey the chemical reaction level to a logic output once a threshold has been reached. Using this method, novel DNA logic functions are derived for nucleotides allowing local digital computations. The thesis also presents business models that enable such technology to be utilised in point of care applications, and experiment as results and business models given for an HIV point of care example are proposed

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

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

Molecular methods for the detection of infectious diseases: bringing diagnostics to the point-of-care

Human infectious diseases represent a leading cause of morbidity and mortality globally, caused by human-infective pathogens such as bacteria, viruses, parasites or fungi. Point-of-care (POC) diagnostics allow accessible, simple, and rapid identification of the organism causing the infection which is crucial for successful prognostic outcomes, clinical management, surveillance and isolation. The research conducted in this thesis aims to investigate novel methods for molecular-based diagnostics. This multidisciplinary project is divided into three main sections: (i) molecular methods for enhanced nucleic acid amplification, (ii) POC technologies, and (iii) sample preparation. The application, design and optimisation of loop-mediated isothermal amplification (LAMP) is investigated from a molecular perspective for the diagnostics of emerging infectious pathogens and antimicrobial resistance. LAMP assays were designed to target pathogens responsible for parasitic (malaria), bacterial and viral (COVID-19) infections, as well as antimicrobial resistance. A novel LAMP-based method for the detection of single nucleotide polymorphisms was developed and applied for diagnostics of antimicrobial resistance, emerging variants and genetic disorders. The method was validated for the detection of artemisinin-resistant malaria. Furthermore, this thesis reports the optimisation of LAMP from a biochemical perspective through the evaluation of its core reagents and the incorporation of enhancing agents to improve its specificity and sensitivity. In order to remove cold-chain storage from the diagnostic workflow, the optimised LAMP protocol was designed to be compatible with lyophilisation. Translation of LAMP to the POC demands the development of detection technologies that are compatible with the advantages offered by isothermal amplification. The use of simple, accessible and portable technologies is investigated in this thesis through the development of: (i) a novel colorimetricLAMP detection method for end-point and low cost detection, and (ii) the combination of LAMP with an electrochemical biosensing platform based on ion-sensitive field effect transistors (ISFETs) fabricated in unmodified complementary metal-oxide semiconductor (CMOS) technology for real-time detection. Lastly, current nucleic acid extraction methods are not transferable to be used outside the laboratory. Research of novel methods for low-cost and electricity-free sample preparation was carried out using cellulose matrices. A novel, rapid (under 10 min) and efficient nucleic acid extraction method from dried blood spots was developed. A sample-to-result POC test requires the implementation and integration of molecular biology, cutting-edge technology and data-driven approaches. The work presented in this thesis aims to set new benchmarks for the detection of infectious diseases at the POC by leveraging on developments in molecular biology and digital technologies.Open Acces

Ion-Sensitive Field-Effect Transistor for Biological Sensing

In recent years there has been great progress in applying FET-type biosensors for highly sensitive biological detection. Among them, the ISFET (ion-sensitive field-effect transistor) is one of the most intriguing approaches in electrical biosensing technology. Here, we review some of the main advances in this field over the past few years, explore its application prospects, and discuss the main issues, approaches, and challenges, with the aim of stimulating a broader interest in developing ISFET-based biosensors and extending their applications for reliable and sensitive analysis of various biomolecules such as DNA, proteins, enzymes, and cells

IonSeq Genome Sequencing

The emergence of advanced DNA sequencing methods has presented disruptive opportunities in biotechnology, establishing the foundation for the personalized medicine industry. Since the completion of the Human Genome Project, the number of genomes sequenced has grown exponentially and the sequencing price has dropped precipitously. To make personalized medicine a reality, there is a need for a large collection of sequenced genomes in order to link specific genes to diseases. IonSeq seeks to be the leading DNA sequencing service, employing new semiconductor- based sequencing technology offered by Ion Torrent, to help pharmaceutical companies generate these libraries of genomes for their drug-development processes. To support sequencing reliability and throughput, IonSeq will explore such technical details such as chip configuration, insertion kinetics, signal generation, base-calling methods, and accuracy metrics. IonSeq will prove a 40 genome/day output, made possible by the massively parallel procedure employed by the sequencers. IonSeq will sequence each genome at a price of $2,000 while the cost of ‘manufacture’ will only be$645. Series A will consist of a $3,682,886 investment and will yield the investors a MIRR of 102.98$4,510,491 and result in a 93.43% MIRR over a three period. The NPV by the time of liquidation or acquisition event will be $39,322,347, at a conservative projected growth rate of 5%

Chemical Current-Conveyor: a new approach in biochemical computation

Biochemical sensors that are low cost, small in size and compatible with integrated circuit technology play an essential part in the drive towards personalised healthcare and the research described in this thesis is concerned with this area of medical instrumentation. A new biochemical measurement system able to sense key properties of biochemical fluids is presented. This new integrated circuit biochemical sensor, called the Chemical Current-Conveyor, uses the ion sensitive field effect transistor as the input sensor combined with the current-conveyor, an analog building-block, to produce a range of measurement systems. The concept of the Chemical Current-Conveyor is presented together with the design and subsequent fabrication of a demonstrator integrated circuit built on conventional 0.35μm CMOS silicon technology. The silicon area of the Chemical Current-Conveyor is (92μm x 172μm) for the N-channel version and (99μm x 165μm) for the P-channel version. Power consumption for the N-channel version is 30μW and 43μW for the P-channel version with a full load of 1MΩ. The maximum sensitivity achieved for pH measurement was 46mV per pH. The potential of the Chemical Current Conveyor as a versatile biochemical integrated circuit, able to produce output information in an appropriate form for direct clinical use has been confirmed by applications including measurement of (i) pH, (ii) buffer index ( ), (iii) urea, (iv) creatinine and (v) urea:creatinine ratio. In all five cases the device has been demonstrated successfully, confirming the validity of the original aim of this research project, namely to produce a versatile and flexible analog circuit for many biochemical measurement applications. Finally, the thesis closes with discussion of another potential application area for the Chemical Current Conveyor and the main contributions can be summarised by the design and development of the first: ISFET based current-conveyor biochemical sensor, called 'Chemical Current Conveyor, CCCII+' has been designed and developed. It is a general purpose biochemical analog building-block for several biochemical measurements. Real-time buffer capacity measurement system, based on the CCCII+, which exploits the imbedded analog computation capability of the CCCII+. Real-time enzyme based CCCII+ namely, Creatinine-CCCII+ and Urea-CCCII+ for real-time monitoring system of renal system. The system can provide outputs of 3 important parameters of the renal system, namely (i) urea concentration, (ii) creatinine concentration, and (ii) urea to creatinine ratio

Metabolomic sensing system for personalised medicine using an integrated CMOS sensor array technology

Precision healthcare, also known as personalised medicine, is based on our understanding of the fundamental building blocks of biological systems, with the ultimate aim to clinically identify the best therapeutic strategy for each individual. Genomics and sequencing technologies have brought this to the foreground by enabling an individual’s entire genome to be mapped for less than a thousand dollar in just one day. Recently, metabolomics, the quantitative measurement of small molecules, has emerged as a field to understand an individual’s molecular profile in terms of both genetics and environmental factors. This is crucial because a genome could only indicate an individual’s susceptibility to a particular disease, whereas a metabolome provides an immediate measurement of body function, enabling a means of diagnosis. However, the current approach of measurements depends on large-scale and expensive equipment such as mass spectroscopy and NMR instrumentation, which does not offer a single analytical platform to detect the entire metabolome. This thesis describes the development of an integrated CMOS sensor array technology as a single platform to quantify different metabolites using specific enzymes. The key stages in the work were: to construct instrumentation systems to perform enzyme assays on the CMOS sensor array; to establish techniques to package the CMOS sensor array for an aqueous environment; to implement and develop a room temperature Ta2O5 sputtering process on CMOS sensor array for hydrogen ion detection; to collaborate with a chemist and investigate an inorganic layer on top of the CMOS ISFET sensor to show an improvement of sensitivity towards potassium ion; to test several different enzyme assays electrochemically and optically and show the functionalities of the sensors; to devise microfluidic channels for segregation of the sensor array into different compartments and perform enzyme immobilisation techniques on CMOS chips; and integrate the packaged chip with microfluidic channels and enzyme immobilisation using 2D inkjet printer into a complete system that has the potential to be used as a multi-enzyme platform for detection of different metabolites. Two CMOS sensor array chips (1) a 256×256-pixel ISFET array chip and (2) a 16×16-pixel Multi-Corder chip were fully understood. Therefore, a high-speed instrumentation system was constructed for the ISFET array chip with a maximum readout speed of 500 frames per second, with 2D and 3D imaging capability, as well as single pixel analysis. Follow by that, a miniaturised measurement platform was implemented for the Multi-Corder chip that has three different sensor arrays, which are ISFET, PD and SPAD. All the sensor arrays can be operated independently or together (ionic sensor and one of the optical sensors). Several post-processing steps were investigated to allow suitable fabrication process on small 4×4 mm2 CMOS chips. Post-processing of the CMOS chips was first established using room temperature sputtering process for Ta2O5 layer, achieving Ta:O ratio of 1:1.77 and a surface roughness of 0.42 nm. This Ta2O5 layer was then fabricated on top of CMOS ISFETs, which improves the ISFET pH sensitivity to 45 mV/pH, with an average drift of 6.5 ± 8.6 mV/hour from chip to chip and a working pH range of 2 to 12. Furthermore, a layer of POMs was drop casted on top of Ta2O5 ISFET to make ISFET sensitive to potassium ions. This was investigated in terms of potassium ions sensitivity, hydrogen ions sensitivity and sodium ions as interfering background ions. The POMs Ta2O5 ISFET was found to have a net potassium sensitivity of 75 mV/pK, with a linear range between pH 1.5 to 3. Moreover, the POMs ISFET has -5 mV/pH in pH sensitivity, showing that it is selectivity towards potassium ions and not hydrogen ions. However, sodium ions were found to produce a large interference towards the pK sensitivity of POMs ISFET and reduced the pK sensitivity of POMs ISFET. Hence, further work is still required to modify POMs layer for better selectivity and sensitivity. Besides that, microfluidic channels were fabricated on top of the CMOS chips that could provide segregation for multiple enzyme assays on a single chip. In addition, a PDMS and a manual dam and fill method were developed to encapsulate the CMOS chips for wet biochemistry measurements. The CMOS sensor array was found to have the ensemble averaging capability to reduce noise as a function of √N , where N is the number of sensors used for averaging. Several enzyme assays that include: hexokinase, lactate dehydrogenase, urease and lipase were tested on the ISFET sensor array. Moreover, using an optical sensor array, namely a PD on the Multi-Corder chip and using LED illumination, quantification of cholesterol levels in human blood serum was demonstrated. Enzyme kinetics calculations were performed for hexokinase and cholesterol oxidase assays and the results were comparable to that obtained from a bench top spectrophotometer. This shows the CMOS sensor array can be used as a low cost portable diagnostic device. Several enzyme immobilisation techniques were explored but were unsuccessful. Alginate enzyme gel immobilisation with a 2D inkjet printer was found to be the best candidate to bio-functionalise the CMOS sensor array. The packaged chip was integrated with microfluidic channels and alginate enzyme gel immobilisation into a complete system, in order to perform an enzyme assay with its control experiments simultaneously on a single chip. As a proof-of-concept, this complete system has the potential to be used as a multiple metabolite quantification platform

Robust low power CMOS methodologies for ISFETs instrumentation

I have developed a robust design methodology in a 0.18 [Mu]m commercial CMOS process to circumvent the performance issues of the integrated Ions Sensitive Field Effect Transistor (ISFET) for pH detection. In circuit design, I have developed frequency domain signal processing, which transforms pH information into a frequency modulated signal. The frequency modulated signal is subsequently digitized and encoded into a bit-stream of data. The architecture of the instrumentation system consists of a) A novel front-end averaging amplifier to interface an array of ISFETs for converting pH into a voltage signal, b) A high linear voltage controlled oscillator for converting the voltage signal into a frequency modulated signal, and c) Digital gates for digitizing and differentiating the frequency modulated signal into an output bit-stream. The output bit stream is indistinguishable to a 1st order sigma delta modulation, whose noise floor is shaped by +20dB/decade. The fabricated instrumentation system has a dimension of 1565 [Mu] m 1565 [Mu] m. The chip responds linearly to the pH in a chemical solution and produces a digital output, with up to an 8-bit accuracy. Most importantly, the fabricated chips do not need any post-CMOS processing for neutralizing any trapped-charged effect, which can modulate on-chip ISFETs’ threshold voltages into atypical values. As compared to other ISFET-related works in the literature, the instrumentation system proposed in this thesis can cope with the mismatched ISFETs on chip for analogue-to-digital conversions. The design methodology is thus very accurate and robust for chemical sensing

Chemical and biochemical sensors based on silicon nanowire field-effect transistor arrays

Field-effect transistors (FETs) made from semiconducting nanowires have great potential as electronic biochemical sensors if they can be integrated as an array in a CMOS-compatible architecture together with microfluidic channels and interfacing electronics. Such nanoscale electronic transducers based on ion-sensitive field-effect transistors could be mass fabricated at reasonable costs. This, in combination with their small size, makes them ideal for personalized medicine and for future implanted sensing devices. The sensing principle is based on adsorption of charged species on the sensor surface, leading to a change in surface potential and subsequently a change in current in the FET channel. Thereby, the high-impedance input signal is transformed into a low-impedance output signal, which is an advantage against classic ion-selective electrodes. The potential for downscaling and integration for the simultaneous detection of multiple parameters make silicon nanowire FETs a promising platform to meet the demand for cheap, multifunctional and scalable sensors. Even though many promising results on chemical and biochemical sensing have been achieved so far, a detailed understanding of the electrolyte surface interaction is still missing. Inconsistent outcomes regarding the effect of electrolyte concentrations and electrical noise, suggest that further quantitative studies are needed. The aspect of the size compatibility between the sensor unit and the analyte species is often emphasized to favor nanoscale FETs. Another aspect, often mentioned, is the surface to volume ratio. Hence smaller sensing units should enhance the sensitivity of the sensor, allowing the detection at ultra-low concentrations or a small number of molecules. Furthermore, the capacitances decrease for smaller sensing units, which could lead to faster response times. However, other aspects such as the intrinsic electronic noise, the analyte diffusion time and surface reaction kinetics have to be considered for the development of an applicable sensor. This thesis was part of a research project aimed at developing a modular, scalable and integrateable sensor platform for the electronic detection of analytes in solution. The main focus lies on the sensor-solution interface and thus the thesis quantitatively compares the experimental data with analytical models. In this work we have established a versatile sensor platform based on silicon nanowire arrays. The sensor functionality was changed by surface modification for the detection of various analytes such as pH, alkaline ions and even FimH proteins. We achieved an ideal pH sensor with a response close to the Nernst limit. Full surface passivation for protons was accomplished for the implementation of a nanoscale reference electrode. Using the differential signal from differently functionalized silicon nanowires we could detect sodium and potassium ions selectively. Ultimately we present the detection of protein-ligand interactions of the physiologically relevant protein FimH. An extended site binding model was derived to calculate the theoretical limits and assess the properties of the surface groups by evaluating the experimental results