Numerical modeling of dielectrophoretic effect for manipulation of bio-particles

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.This text describes different aspects of the design of a Doctor-on-a-Chip device. Doctor-on-a-Chip is a DNA analysis system integrated on a single chip, which should provide all of the advantages that stem from the system integration, such as small sample volume, fast and accurate analysis, and low cost. The text describes all of the steps of the on-chip sample analysis, including DNA extraction from the sample, purification, PCR amplification, novel dielectrophoretic sorting of the DNA molecules, and finally detection. The overview is given of the technologies which are available to make the integration on a single chip possible. The microfluidic technologies that are used to manipulate the sample and other chemical reagents are already known and in this text they are analyzed in terms of their feasibility in the on-chip system integration. These microfluidic technologies include, but are not limited to, microvalves, micromixers, micropumps, and chambers for PCR amplification. The novelty in the DNA analysis brought by Doctor-on-a-Chip is the way in which the different DNA molecules in the sample (for example, human and virus DNA) are sorted into different populations. This is done by means of dielectrophoresis – the force experienced by dielectric particles (such as DNA molecules) when subject to a non-uniform electric field. Different DNA molecules within a sample experience different dielectrophoretic forces within the same electric field, which makes their separation, and therefore detection, possible. In this text, the emphasis is put on numerical modelling of the dielectrophoretic effect on biological particles. The importance of numerical modelling lies in the fact that with the accurate model it is easier to design systems of microelectrodes for dielectrophoretic separation, and tune their sub-micrometre features to achieve the maximum separation efficacy. The numerical model described in this text is also experimentally verified with the novel microelectrodes design for dielectrophoretic separation, which is successfully used to separate the mixture of different particles in the micron and sub-micron range

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Liquid biopsy genotyping in lung cancer: ready for clinical utility?

Liquid biopsy is a blood test that detects evidence of cancer cells or tumor DNA in the circulation. Despite complicated collection methods and the requirement for technique-dependent platforms, it has generated substantial interest due, in part, to its potential to detect driver oncogenes such as epidermal growth factor receptor (EGFR) mutants in lung cancer. This technology is advancing rapidly and is being incorporated into numerous EGFR tyrosine kinase inhibitor (EGFR-TKI) development programs. It appears ready for integration into clinical care. Recent studies have demonstrated that biological fluids such as saliva and urine can also be used for detecting EGFR mutant DNA through application other user-friendly techniques. This review focuses on the clinical application of liquid biopsies to lung cancer genotyping, including EGFR and other targets of genotype-directed therapy and compares multiple platforms used for liquid biopsy

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Micromachines for Dielectrophoresis

An outstanding compilation that reflects the state-of-the art on Dielectrophoresis (DEP) in 2020. Contributions include: - A novel mathematical framework to analyze particle dynamics inside a circular arc microchannel using computational modeling. - A fundamental study of the passive focusing of particles in ratchet microchannels using direct-current DEP. - A novel molecular version of the Clausius-Mossotti factor that bridges the gap between theory and experiments in DEP of proteins. - The use of titanium electrodes to rapidly enrich T. brucei parasites towards a diagnostic assay. - Leveraging induced-charge electrophoresis (ICEP) to control the direction and speed of Janus particles. - An integrated device for the isolation, retrieval, and off-chip recovery of single cells. - Feasibility of using well-established CMOS processes to fabricate DEP devices. - The use of an exponential function to drive electrowetting displays to reduce flicker and improve the static display performance. - A novel waveform to drive electrophoretic displays with improved display quality and reduced flicker intensity. - Review of how combining electrode structures, single or multiple field magnitudes and/or frequencies, as well as variations in the media suspending the particles can improve the sensitivity of DEP-based particle separations. - Improvement of dielectrophoretic particle chromatography (DPC) of latex particles by exploiting differences in both their DEP mobility and their crossover frequencies

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Investigating the Use of Streaming and Robotic Dielectrophoresis to Enable Continuous Cell Sorting and Automatic Cell Transfer in Sample Preparation

The sorting of targeted cells or particles from a sample is a crucial step in the sample preparation process used in medical diagnosis, environmental monitoring, bio-analysis and personalized medicine. Current cell sorting techniques can be broadly classified as label based or label-free. Label-based techniques mostly rely on fluorophores or magnetic nanoparticles functionalized to bind with targeted cells. Although highly specific, this approach can be expensive and suffers from limitations in the availability of suitable markers. Label-free techniques exploit properties inherent to the cell, such as density and size, to simplify the sorting protocol and reduce cost by eliminating the need to incubate samples with labels. However, the specificity of separation is low due to minor differences between the density and size of many cells of interest. In this work, the use of dielectrophoresis (DEP) is emphasized as a label-free technique that exploits the combination of size and membrane capacitance of a cell as a marker. DEP is the movement of dielectric particles in the presence of a non-uniform electric field, which can be towards the electrode (positive DEP) or away from electrode (negative). The cell membrane capacitance used in this project can distinguish between cells based on their type, age, fate, and circadian rhythm to provide higher specificity than other label free sorting techniques. DEP has been demonstrated to separate various bio particles including viruses, plant and animal cells, biomolecules as well as stem cells. The work presented here integrates fabrication, numerical simulations, analysis and experimentation to focus on three main objectives 1) addressing the gaps of knowledge in electrode fabrication 2) developing analytical system for rapid cell sorting of cell population 3) demonstrating feasibility of automated single cell sorting. These are addressed in the following paragraphs in that order. Carbon electrodes are excellent alternatives for DEP because of the ease of fabrication of 3D electrode geometries and low voltages involved. Previous works have used these electrodes for applications like DEP, electrochemical bio sensing applications, fuel cells and micro-capacitors. The fabrication process involves photo patterning of SU-8 posts followed by carbonization in an inert atmosphere. During pyrolysis, the structures retain their shape, but show shrinkage. Though the fabrication process is reproducible, limited knowledge is available about the shrinkage process. Shrinkage affects the design of devices where these structures are used because the electrode dimensions after pyrolysis vary from the design and resulting electric field in the domain is affected. Previous works observed dependence of shrinkage on structure height and width, but a defining relation between shrinkage and the geometry was lacking. In this work, shrinkage is studied as an effect of degassing through the lateral and top surface area of the electrodes. Empirical relations are to enable prediction of shrinkage in the design stage StreamingDEP refers to focusing particles into narrow streams with a proper play of positive DEP and drag force. This is important in continuous sorting because of the high throughput, limited exposure of cells to electric field and ability of integration to further analysis steps. Though streamingDEP has been demonstrated previously, the dependence of the particle focusing on system parameters has not been studied. In this work, an analytical model is built to study the effect of electrode geometry, flow and electric field parameters as well as cell properties. The analytical expression developed here is validated by experiments and simulations. Robotic transfer is required for efficient handling of cells and integration with analysis steps. Liquid handling robots are currently used in laboratories to transfer cells between different steps. Though they have precise control over the transfer of cells, the sorting ability is limited. To address these limitations, a proof-of-concept of roboticDEP device was innovated to enable transfer of targeted cells. The development of this system required studying the influence of DEP parameters in pick up and transfer of cells. The device was studied for elimination of contamination by using flow and electric field. The robotic DEP platform demonstrates a novel and unique approach to automated cell sorting with potential applications for single cell analysis, cell sorting and cell patterning