The Phage Toolbox: Automating Phage Discovery Using Novel Software, Devices, and High-Throughput Methodology

With reports of antibiotic resistance on the rise, the need for alternative therapeutic options is of high importance. Bacteriophage (or phage) therapy is a valuable alternative strategy that holds high promise for treating bacterial infections not responsive to traditional antibiotics. Furthermore, the ability to automate phage therapy research would push the field forward by speeding the time for development, discovery, and reproducible outcomes. This dissertation provides solutions to these challenges by delivering a range of software and device-related tools to streamline and automate phage research. Together, these tools converge to create a &ldquo;phage toolbox&rdquo;, where each tool can be used independently or collectively, paving the way for more automated solutions in phage discovery. The tools presented in this dissertation include (1) PhageBox, (2) PhageFilter, (3) PhageScanner, and (4) DebruijnExtend. PhageBox is an open-source digital microfluidics extension that offers magnetic and temperature control. Furthermore, the proof-of-concept applications show specialized utility towards bacteriophages. PhageFilter is an ultralow memory software tool that introduces a Genome Sequence Bloom Tree (gSBT) to assign short or long-read sequencing data to genomes, enabling accurate DNA filtering, binning, and abundance estimation. Potentially using these filtered sequences, PhageScanner is a machine learning pipeline and software tool that predicts phage virion proteins (PVPs) from metagenomic sequencing data. This extends upon preceding work in this area by using both oligonucleotide and protein sequence information. Lastly, DebruijnExtend presents a tool and algorithm for predicting the secondary structure of proteins from the primary sequence, which can assist with feature extraction and determining subtle differences in the secondary structures of phage structural proteins. The collection of tools presented here offers a comprehensive toolbox for automating phage research from a multidisciplinary perspective, including microfluidics, wet lab biology, and bioinformatics. As we witness the rise of the Internet of Things, Artificial Intelligence, and Synthetic Biology, we are beginning to realize the potential for making significant advances in biology through a multidisciplinary approach. These tools contribute to this paradigm shift by introducing opensource projects that leverage concepts from each of these areas. We envision a future where a consortium of companies and researchers will concentrate on developing bioinformatics software and open-source devices to automate phage biology.</p

A portable metabolomics-on-CMOS platform for point-of-care testing

Metabolomics is the study of the metabolites, small molecules produced during the metabolism. Metabolite levels mirror the health status of an individual and therefore have enormous potential in medical point-of-care (POC) applications. POC platforms are miniaturised and portable systems integrating all steps from sample collection to result of a medical test. POC devices offer the possibility to reduce the diagnostic costs, shorten the testing time, and, ultimately, save lives for several applications. The glucose meter, arguably the most successful example of metabolomics POC platform, has already demonstrated the dramatic impact that such platforms can have on the society. Nevertheless, other relevant metabolomic tests are still relegated to centralised laboratories and bulky equipment. In this work, a metabolomics POC platform for multi-metabolite quantification was developed. The platform aims to untap metabolomics for the general population. As case studies, the platform was designed and evaluated for prostate cancer and ischemic stroke. For prostate cancer, new affordable diagnostic tools to be used in conjunction with the current clinical standard have are needed to reduce the medical costs due to overdiagnosis and increase the survival rate. Thus, a novel potential metabolic test based on L-type amino acids (LAA) profile, glutamate, choline, and sarcosine blood concentrations was developed. For ischemic stroke, where the portable and rapid test can make a difference between life and death, lactate and creatinine blood levels were chosen as potential biomarkers. All the target metabolites were quantified using an optical method (colorimetry). The platform is composed of three units: the cartridge, the reader, and the graphical user interface (GUI). The cartridge is the core of the platform. It integrates a CMOS 16x16 array of photodiodes, capillary microfluidics, and biological receptors onto the same ceramic package. To measure multiple metabolites, a novel method involving a combination of replica moulding and injection moulding was developed for the monolithic integration of microfluidics onto integrated chips. The reader is composed of a custom PCB and a microcontroller board. It is used for addressing, data digitisation and data transfer to the GUI. The GUI - a software running on a portable electronic device - is used for interfacing the system, visualise, acquire, process, and store the data. The analysis of the microfluidic structures showed successful integration. The selection of the specific chemistry for detecting the analytes of interest was demonstrated to be suitable for the performance of the sensors. Quick and reliably capillary flow of human plasma, serum and blood was demonstrated. On-chip quantification of the target metabolites was demonstrated in diluted human serum and human plasma. Calibration curves, kinetics parameter and other relevant metrics were determined. For all the metabolites, the limits of detection were lower than the physiological range, demonstrating the capability of the platform to be used in the target applications. Multi-metabolite testing capability was also demonstrated using commercially and clinically sourced human plasma. For multiplexed assays, reagents were preloaded in the microfluidic channel and lyophilised. Lyophilisation also improved the shelf-life of the reagents. Alternative configurations, involving the use of paper microfluidics, integration of passive blood filter and use of whole blood, were investigated. The chracterisation of the platform culminated with a clinical evaluation for both the target applications. The same platform with minimal modification of the cartridge was able to provide clinically relevant information for both the distinct applications, highlighting the versatility of the platform for POC determination of metabolic biomarkers. For prostate cancer, the platform was used for the quantification of the potential metabolic biomarker in 10 healthy samples and 16 patients affected by prostate cancer. LAA, glutamate and choline average concentrations were elevated in the cancer group with respect to the control group and were therefore regarded as metabolic biomarkers in this population. Metabolomic profiles were used to train a classifier algorithm, which improved the performance of the current clinical blood test, for this population. For ischemic stroke, lactate determination was performed in clinically sourced samples. Clinical evaluation for ischemic stroke was performed using 10 samples from people diagnosed with ischemic stroke. Results showed that the developed platform provided comparable results with an NHS-based gold standard method in this population. This comparison demonstrated the potential of the platform for its on-the-spot use. The developed platform has the potential to lead the way to a new generation of low-cost and rapid POC devices for the early and improved diagnosis of deadly diseases

A Modular design framework for Lab-On-a-Chips

This research discusses the modular design framework for designing Lab-On-a-Chip (LoC) devices. This work will help researchers to be able to focus on their research strengths, without needing to learn details of LoCs design, and they can reuse existing LoC designs

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

Design of electronic systems for automotive sensor conditioning

This thesis deals with the development of sensor systems for automotive, mainly targeting the exploitation of the new generation of Micro Electro-Mechanical Sensors (MEMS), which achieve a dramatic reduction of area and power consumption but at the same time require more complexity in the sensor conditioning interface. Several issues concerning the development of automotive ASICs are presented, together with an overview of automotive electronics market and its main sensor applications. The state of the art for sensor interfaces design (the generic sensor interface concept), consists in sharing the same electronics among similar sensor applications, thus saving cost and time-to-market but also implementing a sub-optimal system with area and power overheads. A Platform Based Design methodology is proposed to overcome the limitations of generic sensor interfaces, by keeping the platform generality at the highest design layers and pursuing the maximum optimization and performances in the platform customization for a specific sensor. A complete design flow is presented (up to the ASIC implementation for gyro sensor conditioning), together with examples regarding IP development for reuse and low power optimization of third party designs. A further evolution of Platform Based Design has been achieved by means of implementation into silicon of the ISIF (Intelligent Sensor InterFace) platform. ISIF is a highly programmable mixed-signal chip which allows a substantial reduction of design space exploration time, as it can implement in a short time a wide class of sensor conditioning architectures. Thus it lets the designers evaluate directly on silicon the impact of different architectural choices, as well as perform feasibility studies, sensor evaluations and accurate estimation of the resulting dedicated ASIC performances. Several case studies regarding fast prototyping possibilities with ISIF are presented: a magneto-resistive position sensor, a biosensor (which produces pA currents in presence of surface chemical reactions) and two capacitive inertial sensors, a gyro and a low-g YZ accelerometer. The accelerometer interface has also been implemented in miniboards of about 3 cm2 (with ISIF and sensor dies bonded together) and a series of automatic trimming and characterization procedures have been developed in order to evaluate sensor and interface behaviour over the automotive temperature range, providing a valuable feedback for the implementation of a dedicated accelerometer interface

Novel pneumatic circuit for the computational control of soft robots

Soft robots are of significant research interest in recent decades due to their adaptability to unstructured environments and safe interaction with humans. Soft pneumatic robots, one of the most dominant subsets of soft robots, utilize the interaction between soft elastomeric materials and pressurized air to achieve desired functions. However, the systems currently used for signal computation and pneumatic regulation often make use of rigid valves, pumps, syringe drivers, microcontrollers et al. These bulky and non-integrable devices limit the performance of pneumatically-driven soft robots, carrying challenges for the robot to be miniaturized, untethered, and agile. This DPhil aims to develop pneumatic circuits that can be integrated into the soft robot bodies while performing both onboard computation and control. This thesis presents our contributions towards the aforementioned objective step by step. Firstly, we designed a 3D-printable bistable valve with tunable behaviours for controlling soft pneumatic robots. As an integrable control device, the valve stores one bit of binary information without requiring a constant energy supply and correspondingly controls a pneumatic chamber. Secondly, in order to reduce the number of valves required to control multi-chamber soft robots, we introduced a modular approach to design multi-channel bistable valves based on the previous work. Thirdly, in order to achieve continuous pressure modulation with integrable devices, we designed a soft proportional valve, utilizing the continuous deformation of Magnetorheological Elastomer (MRE) under magnetic flux. Apart from the analogue activation manner, this design also ensures a fast response time, operating at a time scale of tens of milliseconds, much shorter than the mechanical response time of most soft pneumatic actuators. Fourthly, to achieve onboard proportional control of multi-chamber soft robots, we developed an MRE valve array with an embedded cooling chamber. Physical experiments showed that our MRE valve array ensured the independence and accuracy of each valve unit within it, with a significantly lowered temperature of 73.9 $^o$C under 5 minutes of operation. Lastly, we developed an open-source software toolbox supporting the design of integrable pneumatic logic circuits to enhance their accessibility and performance. The toolbox comes with a graphical user interface (GUI) to take users' desired logic functions in the form of a truth table and a set of 2D space constraints related to the available space onboard the robot. It then schedules the pneumatic circuit which performs the desired computation within the space constraints and produces a 3D-printable CAD file that can be fabricated and used directly. The work presented in this thesis enables the community to simplify the process of integrating control devices into soft pneumatic robots, thereby paving the way for a new generation of fully untethered and autonomous soft robots

SUITE an Innovative Bioreactor Platform for in vitro Experiments

In-vitro cell cultures are a fundamental step in preclinical drug testing and are of great interest to the pharmaceutical industry. The most common method for culturing cells is in cell culture incubators. These are large and cumbersome and all mechanical stimuli are absent. They are nevertheless used ubiquitously and their results quoted as "standards" of in-vitro protocols. Several alternative culture methods have been proposed, and many systems are currently available commercially. Indeed, systems and devices for maintaining cells and tissues in controlled physical conditions, or bioreactors, have become an important tool in many areas of research. This is not only due to the growing interest in tissue engineering but also because it is now being increasingly recognised that cells respond not only to their biochemical, but also to their physical environment, and both cues are necessary to create a biomimetic habitat. However most bioreactors for cell culture and tissue engineering are cumbersome and only provide a few cues such as flow or strain, allowing limited control and flexibility. Since drug testing involves a large number of tests on identical cell cultures, a single well culture is inadequate and costly both in time and money. The High Throughput Screening (HTS), is a methodology for scientific experimentation widely used in drug discovery, based on a brute-force approach to collect a large amount of experimental data in less time and using less animals. The parallel nature of HTS makes it possible to collect a large amount of data from a small number of experiments and in a very short time. HTS, however, suffers from a significant problem that may affect the relevance of tests: the environment discrepancy problem. Another problem related with the actual drug testing and tissue engineering experiments is the enormous number of animals that have to be scarified every year. The aim of this study was to develop a generic platform or SUITE (Supervising Unit for In-vitro TEsting) for cell, tissue and organ culture composed of two main components: a universal control unit and an array of bioreactor chambers. The platform provides a biomimetic habitat to cells and tissues since the environment in the chambers is controlled and regulated to provide biomechanical and biophysical stimuli similar to those found in-vivo. In this work I describe how a new concept of cell culture bioreactor was developed by integrating different technologies and research fields. The data extracted using this new cell culture approach is more predictive of the in vivo response with respect to the multi-well approach, particularly for drug related studies. The starting point was a thorough analysis of currently used in-vitro methods; their pros and cons were assessed to exploit their advantages and overcome or circumvent their disadvantages. As far as the culture chamber is concerned, the approach was to use the methods and materials commonly employed in microfluidic fabrication, but at scales compatible with classical culture systems such as petri-dishes and multiwells. This renders the bioreactors more amenable to use by biologists and enables the use of cell densities comparable with classic systems as well as the use of conventional assaying techniques. In most cases, the cell culture chambers are thus made out of PDMS (Polydimethylsiloxane), using soft-moulding with micro- or mini-machined masters, or what we call Soft Milli-molding. A system on a plate Multi Compartmental Modular Bioreactor (MCmB) was developed using this technology. The MCmB is a modular chamber for high throughput multi compartmental bioreactor experiments. It is designed to be used in a wide range of applications and with various cell types. A precise stimulus application is also very important to better understand the correlation between physical variables and pathologies allowing a more accurate study of the tissue physiology and pathologies. For this reason in these thesis three additional stimulation chambers for vascular and articular cartilage stimulation respectively were also designed and tested. The control system was developed to be user-friendly, flexible and expandable to include new stimuli and was based on modular components, including motors and sensors. Importantly a single software interface was designed to allow data acquisition and monitoring of several chambers in series or in parallel. Using SUITE, high throughput experiments can be performed in an in vivo-like simulated environment for a long time to simulate different physiological or pathological scenarios or for toxicity testing of cells, tissues or in-vitro organ models

Solid State Circuits Technologies

The evolution of solid-state circuit technology has a long history within a relatively short period of time. This technology has lead to the modern information society that connects us and tools, a large market, and many types of products and applications. The solid-state circuit technology continuously evolves via breakthroughs and improvements every year. This book is devoted to review and present novel approaches for some of the main issues involved in this exciting and vigorous technology. The book is composed of 22 chapters, written by authors coming from 30 different institutions located in 12 different countries throughout the Americas, Asia and Europe. Thus, reflecting the wide international contribution to the book. The broad range of subjects presented in the book offers a general overview of the main issues in modern solid-state circuit technology. Furthermore, the book offers an in depth analysis on specific subjects for specialists. We believe the book is of great scientific and educational value for many readers. I am profoundly indebted to the support provided by all of those involved in the work. First and foremost I would like to acknowledge and thank the authors who worked hard and generously agreed to share their results and knowledge. Second I would like to express my gratitude to the Intech team that invited me to edit the book and give me their full support and a fruitful experience while working together to combine this book

VLSI design concepts for iterative algorithms

Circuit design becomes more and more complicated, especially when the Very Large Scale Integration (VLSI) manufacturing technology node keeps shrinking down to nanoscale level. New challenges come up such as an increasing gap between the design productivity and the Moore’s Law. Leakage power becomes a major factor of the power consumption and traditional shared bus transmission is the critical bottleneck in the billion transistors Multi-Processor System–on–Chip (MPSoC) designs. These issues lead us to discuss the impact on the design of iterative algorithms. This thesis presents several strategies that satisfy various design con- straints, which can be used to explore superior solutions for the circuit design of iterative algorithms. Four selected examples of iterative al- gorithms are elaborated in this respect: hardware implementation of COordinate Rotation DIgital Computer (CORDIC) processor for sig- nal processing, configurable DCT and integer transformations based CORDIC algorithm for image/video compression, parallel Jacobi Eigen- value Decomposition (EVD) method with arbitrary iterations for com- munication, and acceleration of parallel Sparse Matrix–Vector Multipli- cation (SMVM) operations based Network–on–Chip (NoC) for solving systems of linear equations. These four applications of iterative meth- ods have been chosen since they cover a wide area of current signal processing tasks. Each method has its own unique design criteria when it comes to the direct implementation on the circuit level. Therefore, a balanced solution between various design tradeoffs is elaborated for each method. These tradeoffs are between throughput and power consumption, com- putational complexity and transformation accuracy, the number of in- ner/outer iterations and energy consumption, data structure and net- work topology. It is shown that all of these algorithms can be imple- mented on FPGA devices or as ASICs efficiently

An open-source compiler and PCB synthesis tool for digital microfluidic biochips

This paper describes a publicly available, open source software framework designed to support research efforts on algorithms and control for digital microfluidic biochips (DMFBs), an emerging laboratory-on-a-chip (LoC) technology. The framework consists of two parts: a compiler, which converts an assay, specified using the BioCoder language, into a sequence of electrode activations that execute out the assay on the DMFB; and a printed circuit board (PCB) layout tool, which includes algorithms to reduce the number of control pins and PCB layers required to drive the chip from an external source. The framework also includes a suite of visualization tools for debugging, and a collection of front-end algorithms that generate mixing/dilution trees for sample preparation