Design and Operation of a Microwave Flow Cytometer for Single Cell Detection and Identification

Microwave dielectric sensing has become a popular technique in biological cell sensing for its potential in online, label-free, and real-time sensing. At microwave frequencies probing signals are sensitive to intracellular properties since they are able to penetrate cell membranes, making microwave flow cytometry a promising technology for label-free biosensing. In this dissertation a microwave flow cytometer is designed and used to measure single biological cells and micro particles. A radio frequency (RF)/microwave interferometer serves as the measurement system for its high sensitivity and tunability and we show that a two-stage interferometer can achieve up to 20 times higher sensitivity than a single interferometer. A microstrip sensor with an etched microfluidic channel is used as the sensing structure for measuring single cells and particles in flow. The microwave flow cytometer was used to measure changes in complex permittivity, , of viable and nonviable Saccharomyces cerevisiae and Saccharomyces pastorianus yeast cells and changes in complex permittivity and impedance of two lifecycle stages of Trypanosoma brucei, a unicellular eukaryotic parasite found in sub-Saharan Africa, at multiple frequencies from 265 MHz to 7.65 GHz. Yeast cell measurements showed that there are frequency dependent permittivity differences between yeast species as well as viability states. Quadratic discriminate analysis (QDA) and k-nearest neighbors (KNN) were employed to validate the ability to classify yeast species and viability, with minimum cross-validation error of with cross validation errors of 19% and 15% at 2.38 GHz and 265 MHz, respectively. Measurements of changes in permittivity and impedance of single procyclic form (PCF) and bloodstream form (BSF) T. brucei parasites also showed frequency dependence. The two cell forms had a strong dependence on the imaginary part of permittivity at 2.38 GHz and below and a strong dependence on the real part of permittivity at 5.55 GHz and above. Three PCF cell lines were tested to verify that the differences between the two cell forms were independent of cell strain. QDA gave maximum cross-validation errors of 15.4% and 10% when using one and three PCF strains, respectively. Impedance measurements were used to improve cell classification in cases where the permittivity of a cell cannot be detected. Lastly, a microwave resistance temperature detector (RTD) is designed, and a model is developed to extract the temperature and complex permittivity of liquids in a microfluidic channel. The microwave RTD is capable of measuring temperature to within 0.1°C. The design can easily be modified to increase sensitivity be lengthening the sensing electrode or modified for smaller volumes of solute by shortening the electrode

Measurement and mathematical modeling of hyperthermia induced bioeffects in pancreatic cancer cells

Doctor of PhilosophyDepartment of Electrical and Computer EngineeringPunit PrakashSurgical resection is the standard of care for pancreatic cancer, although treatment outcomes remain poor, and a large fraction of the patient population are not surgical candidates. Minimally invasive interventions employing non-ionizing energy, such as image-guided thermal ablation, are under investigation for treatment of unresectable tumors and potentially for debulking and downstaging tumors. Tissue regions at the periphery of an ablation zone are exposed to sub-ablative thermal profiles (referred to as “mild hyperthermia”), which may induce a range of bioeffects including change in perfusion, immune modulation, and others. Bioeffects induced by heating are a function of intensity of heating and duration of thermal exposure. This dissertation presents a suite of tools for integrated in vitro experimental studies and modeling for characterizing bioeffects following thermal exposure to pancreatic cancer cells. An instrumentation platform was developed for exposing monolayer cell cultures to temperatures in the range 42–50°C for 3–60 minutes. The platform was employed to determine the Arrhenius kinetic parameters of thermal injury to pancreatic cancer cells (i.e. loss in viability) following heating. When coupled with bioheat transfer models, these parameters facilitate investigations of thermal injury profiles in pancreatic tumors following thermal exposure with practical devices. There has been growing interest in exploring the potential of thermal therapies for modulating tumor—immune system interactions, due in part to release of damage associated molecular patterns (DAMPs) from stressed tumor cells and their role in recruiting and activating antigen presenting cells. The in vitro thermal exposure platform was further expanded to allow for experimental measurement of extracellular DAMPs released from murine pancreatic cancer cells following heating to temperatures in the range 42 – 50°C for 3-60 mins. A model predicting the dynamics of heat-induced DAMPs release was developed and may inform the design of experiments investigating the role of heat in modulating the anti-tumor immune response. While in vitro experiments on monolayers are informative, 3D cell cultures (e.g., spheroid, organoids) provide an experimental platform accommodating multiple cell types in an environment that may be more representative of tumors in vivo. Furthermore, while the water-bath based in vitro platform applied for monolayers is well suited to achieving near-uniform temperature profiles, in vivo delivery of hyperthermia often yields a gradient of temperatures that is not achieved through water-bath based heating. Thus, an in vitro platform for exposing cells in 3D culture (co-culture of multiple cell populations) to 2.45 GHz microwave hyperthermia was developed. The platform includes a printed patch antenna and associated thermal management elements and was applied to study changes in gene expression profile of a 3D culture of pancreatic cancer cells and fibroblasts. This non-contact microwave heating approach may help enable additional studies for exploring the bioeffects of heat on cancer cells

Early warning generation for process with unknown disturbance

Process safety has paramount importance in a chemical process. A well designed control system is the first layer in a process system. The warning system works as the upper protection layer above the control system. It alerts the operators when the control system fails to prevent an undesired situation. A typical warning system issues warnings when a monitored variable exceeds the threshold. Often these do not allow operators sufficient lead-time to take corrective actions. With the motivation of improving the operator’s working environment by providing lead-time, the current research develops a predictive warning scheme using a moving horizon technique. The main hypothesis proposed in this thesis is given the current state of process system, the future states of the system can be predicted using a suitable model of process system. If an external input disturbs the system state, the controller will try to bring the system within the desired control/safety limits of the system. A warning is issued if it is determined that the control system will not be able to keep the system withing the safety limits. Based on the hypothesis, warning systems were developed for both linear and nonlinear systems. For linear systems, using the gain of the models, a linear constrained optimization problem was formulated. Linear programming (LP) was used to determine if the system will remain within the safety limits or not. In case the LP determines that there is no feasible solution within the constrained limits, warnings are issued. The predictive warning scheme was also extended for nonlinear systems. A non-linear receding horizon predictor was used to predict the future states of the nonlinear system. However, for nonlinear system formulation leads to nonlinear constrained optimization problem, where the constraints are the safety limits. Controller’s ability to keep the predicted states inside the safety limit was checked using a feasibility test algorithm. The algorithm uses a constraint separation method with weighting functions to determine the existence of a feasible solution. The algorithm calculates the global minimum of the objective function. If the global minimum of the objective function is positive, it signifies no feasible solution within the input and output constraints of the system and a warning is issued. Prediction of the effect of the disturbances requires the knowledge of the disturbances. In process industries, disturbances are often unmeasured. This thesis also investigates the estimation of unknown disturbances. An iterative Expectation Minimization (EM) algorithm was proposed for the estimation of the unknown states and disturbances of nonlinear systems. Efficacy of the proposed methods was shown through a number of case studies. The warning system for the linear system was simulated on a virtual plant of a continuous stirred tank heater (CSTH). The nonlinear warning system was implemented on a continuous stirred tank reactor (CSTR). Both case studies showed that, the proposed method was capable of providing a warning earlier than the traditional methods that issues warning based on the measured signals

Automating Gene Editing Using Digital Microfluidics to Decipher Cancer Pathways

Gene-editing techniques such as RNA-guided endonuclease systems are becoming increasingly popular for phenotypic screening. Such screens are normally conducted in arrayed or pooled formats. There has been considerable interest in recent years to find new technological methods for conducting these gene-editing assays. We report here the first digital microfluidic method that can automate arrayed gene-editing in mammalian cells. Functional microfluidic devices were designed and optimized to produce repeatable experiments and validate the relevant biological processes on device. Specifically, this method was useful in culturing lung cancer cells for up to six days, as well as implementing automated gene transfection and knockout procedures. In addition, a standardized imaging pipeline to analyse fluorescently labelled cells was also designed and implemented during these procedures. A gene editing assay for interrogating the MAPK/ERK pathway was performed to show the utility of our platform and to determine the effects of knocking out the RAF1 gene in lung cancer cells. In addition to gene knockout, we also treated the cells with an inhibitor, Sorafenib Tosylate, to determine the effects of enzymatic inhibition. The combination of enzymatic inhibition and guide targeting on device resulted in lower drug concentrations for achieving half-inhibitory effects (IC50) compared to cells treated only with the inhibitor, confirming that lung cancer cells are being successfully edited on the device. We propose that this system will be useful for other types of gene-editing assays and applications related to personalized medicine

Characterising fitness landscapes of protein evolution by next-generation sequencing

A protein’s amino acid sequence determines its structural, chemical and physical properties, yet how sequence variation influences protein function is still incompletely understood. Protein fitness landscapes powerfully describe the sequence-function relationship by dividing sequence space into functional hills and valleys. This representation is often invoked yet lacks experimental evidence; the immense vastness of possible sequence space makes comprehensive high-quality datasets difficult to obtain. Laboratory directed evolution has focused on optimal utilisation of substitution libraries, however examination of functional innovation in Nature shows that short insertions and deletions (InDels) also play a key role. Beyond rare targeted studies of specific InDels, high-throughput data on fitness landscape for mutations other than substitutions are lacking entirely. In my PhD, I worked towards experimentally describing the fitness landscapes of InDels and substitutions in three systems: GFP, phosphotriesterase (PTE) and the kinase MKK1 docking domain. Towards this goal, I established two experimental assays (GFP, PTE) for deep mutational scanning and a new software toolkit, InDelScanner, for interpreting resulting data that contain InDels. With GFP, I sorted the deletions and substitution libraries into three activity fractions using FACS, then deep sequenced them with Illumina MiSeq to obtain a pilot dataset. The comparison of deletion effects between different lengths of deletions (-3, -6 and -9 bp) indicates that deletions are partially tolerated in eGFP, with tolerance improved for short deletions and in the stabilised starting point GFP8. Further interpretation of data was complicated by limited resolution in the sequencing dataset stemming from poor FACS separation, so I optimised the conditions for better sorting resolution using the mKate2 fluorescent protein as an expression reporter. In the second iteration of the activity sorting I additionally included UMIs in the plasmid design to improve the utilisation of NGS capacity. In the case of PTE, I performed proof-of-concept experiments for microfluidic droplet sorting in an integrated device with an in-line incubation line and a fluorescent sorting design. In parallel, testing of solubility and activity of random InDel variants showed that functional InDels do not necessarily suffer from a stability handicap, making InDel mutagenesis a viable strategy for gene randomisation in directed evolution. One challenge of InDel library data analysis is that InDels are not compatible with existing, substitution-focused software. Using the GFP deletions dataset, I developed the InDelScanner scripts which accurately detect, aggregate and filter insertions, deletions and substitutions. Using the scripts for composition analysis of TRIAD libraries in PTE showed these libraries are well balanced and highly diverse. Finally, I used the InDelScanner scripts to interpret a deep mutational scanning dataset that recorded the sequence preferences in the MKK1 docking domain, acting to activate ERK2. This experiment showed that the fitness landscape in this kinase pair is shaped by the activating effect of hydrophobic residues in the docking groove, as well as widespread positive epistasis. Together, the projects in this thesis demonstrate that deep mutational scanning experiments are a powerful method for exploring the sequence-function relationship in proteins, which can extend into comparison of different types of mutations as well as probing their (epistatic) interactions.BBSRC BB/M011194/

LARGE TARGET TISSUE NECROSIS OF RADIOFREQUENCY ABLATION USING MATHEMATICAL MODELLING

Radiofrequency ablation (RFA) is a clinic tool for the treatment of various target tissues. However, one of the major limitations with RFA is the ‘small’ size of target tissues that can be effectively ablated. By small it is meant the size of the target tissue is less than 3 cm in diameter of the tissue otherwise ‘large’ size of tissue in this thesis. A typical problem with RFA for large target tissue is the incompleteness of tumour ablation, which is an important reason for tumour recurring. It is widely agreed that two reasons are responsible for the tumour recurring: (1) the tissue charring and (2) the ‘heat-sink’ effect of large blood vessels (i.e. ≥3 mm in diameter). This thesis study was motivated to more quantitatively understand tissue charring during the RFA procedure and to develop solutions to increase the size of target tissues to be ablated. The thesis study mainly performed three tasks: (1) evaluation of the existing devices and protocols to give a clear understanding of the state of arts of RFA devices in clinic, (2) development of an accurate mathematical model for the RFA procedure to enable a more quantitative understanding of the small target tissue size problem, and (3) development of a new protocol based on the existing device to increase the size of target tissues to be ablated based on the knowledge acquired from (1) and (2). In (1), a design theory called axiomatic design theory (ADT) was applied in order to make the evaluation more objective. In (2), a two-compartment finite element model was developed and verified with in vitro experiments, where liver tissue was taken and a custom-made RFA system was employed; after that, three most commonly used internally cooled RFA systems (constant, pulsed, and temperature-controlled) were employed to demonstrate the maximum size of tumour that can be ablated. In (3) a novel feedback temperature-controlled RFA protocol was proposed to overcome the small target tissue size problem, which includes (a) the judicious selection of control areas and target control temperatures and (b) the use of the tissue temperature instead of electrode tip temperature as a feedback for control. The conclusions that can be drawn from this thesis are given as follows: (1) the decoupled design in the current RFA systems can be a critical reason for the incomplete target tissue necrosis (TTN), (2) using both the constant RFA and pulsed RFA, the largest TTN can be achieved at the maximum voltage applied (MVA) without the roll-off occurrence. Furthermore, the largest TTN sizes for both constant RFA and pulsed RFA are all less than 3 cm in diameter, (3) for target tissues of different sizes, the MVA without the roll-off occurrence is different and it decreases with increase of the target tissue size, (4) the largest TTN achieved by using temperature-controlled RFA under the current commercial protocol is still smaller 3 cm in diameter, and (5) the TTN with and over 3 cm in diameter can be obtained by using temperature-controlled RFA under a new protocol developed in this thesis study, in which the temperature of target tissue around the middle part of electrode is controlled at 90 ℃ for a standard ablation time (i.e. 720 s). There are a couple of contributions with this thesis. First, the underlying reason of the incomplete TTN of the current commercially available RFA systems was found, which is their inadequate design (i.e. decoupled design). This will help to give a guideline in RFA device design or improvement in the future. Second, the thesis has mathematically proved the empirical conclusion in clinic that the limit size of target tissue using the current RFA systems is 3 cm in diameter. This has advanced our understanding of the limit of the RFA technology in general. Third, the novel protocol proposed by the thesis is promising to increase the size of TTN with RFA technology by about 30%. The new protocol also reveals a very complex thermal control problem in the context of human tissues, and solving this problem effectively gives implication to similar problems in other thermal-based tumour ablation processes

Advancements in Label-free Biosensing Using Field-Effect Transistors and Aided by Molecular Dynamics Simulations

Biosensors are used to characterize or measure concentrations of physiologically or pathologically significant biomarkers that indicate the health status of a patient, for example, a biomarker associated with a specific disease or cancer. Presently, there is a need to improve the capabilities of biosensors, which includes their rate of detection, limit of detection, and usability. With respect to usability, it is advantageous to develop biosensors that can detect a biomarker that is not labeled, such as with a conventional fluorescent, magnetic, or radioactive label, prior to characterization or measurement by that biosensor. Such biosensors are known as label-free biosensors and are the primary focus of this work. Biosensors are principally evaluated by two standards: their sensitivity to detect a target biomarker at physiologically relevant concentrations and their specificity to detect only the target biomarker in the presence of other molecules. The elements of biosensing critical to improving these two standards are: biorecognition of the biomarker, immobilization of the biorecognition element on the biosensor, and transduction of biomarker biorecognition to a measurable signal. Towards the improvement of sensitivity, electrostatically sensitive field-effect transistors (FET) were fabricated in a dual-gate configuration to enable label-free biosensing measurements with both high sensitivity and signal-to-noise ratio (SNR). This high performance, quantified with several metrics, was principally achieved by performing a novel annealing process that improved the quality of the FET’s semiconducting channel. These FETs were gated with either a conventional oxide or an ionic liquid, the latter of which yielded quantum capacitance-limited devices. Both were used to measure the activity of the enzyme cyclin-dependent kinase 5 (Cdk5) indirectly through pH change, where the ionic-liquid gated FETs measured pH changes at a sensitivity of approximately 75 times higher than the conventional sensitivity limit for pH measurements. Lastly, these FETs were also used to detect the presence of the protein streptavidin through immobilization of a streptavidin-binding biomolecule, biotin, to the FET sensing surface. To study the biomolecular factors that govern the specificity of biomarker biorecognition in label-free biosensing, molecular dynamics (MD) simulations were performed on several proteins. MD simulations were first performed on the serotonin receptor and ion channel, 5-HT3A. These simulations, which were performed for an order of magnitude longer than any previous study, demonstrate the dynamic nature of serotonin (5-HT) binding with 5-HT3A. These simulations also demonstrate the importance of using complex lipid membranes to immobilize 5-HT3A for biosensing applications to adequately replicate native protein function. The importance of lipid composition was further demonstrated using MD simulations of the ion channel alpha-hemolysin (αHL). The results of these simulations clearly demonstrate the lipid-protein structure-function relationship that regulates the ionic current though a lipid membrane-spanning ion channel. Finally, to demonstrate the impact of MD simulations to inform the design of FET biosensing, a strategy to use FETs to measure the ultra-low ionic currents through the ion channel 5-HT3A is outlined. This strategy leverages critical elements of 5-HT biorecognition and ion channel immobilization extracted from MD simulations for the design of the proposed FET sensing surface interface

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

Selected Papers from the 1st International Electronic Conference on Biosensors (IECB 2020)

The scope of this Special Issue is to collect some of the contributions to the First International Electronic Conference on Biosensors, which was held to bring together well-known experts currently working in biosensor technologies from around the globe, and to provide an online forum for presenting and discussing new results. The world of biosensors is definitively a versatile and universally applicable one, as demonstrated by the wide range of topics which were addressed at the Conference, such as: bioengineered and biomimetic receptors; microfluidics for biosensing; biosensors for emergency situations; nanotechnologies and nanomaterials for biosensors; intra- and extracellular biosensing; and advanced applications in clinical, environmental, food safety, and cultural heritage fields

Abstracts of Papers Presented at the 2005 Pittsburgh Conference

To attend or not to attend, that is the question. The Pittsburgh Conference continues to pose this conundrum to conferees and exhibitors alike. This year's conference was the first to be presented without a set of paper abstracts—a good thing some would say but this old codger always used the paper abstracts to select papers of interest to our readership and to seek a full publication. The exhibit took its usual format but it seemed that there were less manufacturers present. The information presented to the attendees was also lacking and many companies' details were missing from the final program book, an omission no doubt on their behalf—my company was one of these—however I feel sure that past Pittcon organizers would have been more persistent in getting the required details for the audience. As is now the norm, many of the presentations take the form of posters displayed within the exhibition area. Without a driver to get the audience there, the traffic was slow, to say the least. Lecture presentations were also attended in a mixed fashion. So the Pittsburgh Conference show moves on, and again next year it will be held in Orlando from 12 March to 17 March 2006. No doubt I will be there making it a straight 31 in a row; in Pittsburgh Conference terms I am just a beginner with many of the attendees making more shows in a run than that. Selected abstracts dealing with topics of interest to the readers of this journal follow—hopefully many of these groups will be willing to publish their work either within this journal or elsewhere