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

Cell Separations and Sorting

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.9b05357.NIBIB Grant P41-EB020594COBRE Grant 5P20GM13042

Acoustic Standing Wave Manipulation of Particles and Cells in Microfluidic Chips

The rise of MEMS and µTAS techniques has created a whole new family of microfluidic devices for a wide range of chemical and biomedical analyses to be performed on small Lab-on-a-chip platforms. The operations often include small samples of particle or cell suspensions on which separation, mixing, trapping or sorting is performed. External fields and forces are used for these operations, and this thesis is specifically focused the development of ultrasonic standing wave technology and the use of acoustic force fields to perform bioanalytical unit operations. The combination of acoustic standing waves and the laminar flow in microfluidics has proven to be well suited for performing particle and cell separation. The fundamental acoustic separator used in this thesis consists of a microfluidic flow channel with a three way flow splitter (trifurcation) in the end of the channel. An acoustic standing wave field is applied to the main flow channel by attaching the transducer underneath the chip. The acoustic standing wave is however obtained perpendicular to the axial propagation of the wave field and the direction of the flow. The half wavelength resonance affects rigid particles or cells driving them into the acoustic pressure node while liquid spheres having other density and compressibility properties may move to the pressure antinode. This enables acoustic separation of different particle types. Blood has proven to be very suitable for acoustic cell manipulation. An application where lipid particles can be removed acoustically from shed blood from open heart surgery is demonstrated. An application for acoustic plasmapheresis is also shown where high quality blood plasma is generated. Different separator designs, device material, and the influence of the separation channel cross-section design are also investigated

Microfluidic-integrated vertical electrodes employed in impedance-based cytometry:potential application in immunotherapies

During the last decades, the growing interest for single-cell analysis has led to the creation of a number of microfluidic and lab-on-a chip (LOC) platforms for characterizing cellular samples. In that context label-free based platforms are minimally invasive and offer the notable advantages of reducing alteration of the analyzed sample and granting its re-employment. The study of intrinsic features of single cells independent from markers is commonly attained using electrical and mechanical-based techniques. Electrical-based techniques have been widely employed in LOC applications, both for characterizing and for manipulating cell samples. The translation of these approaches to single-cells necessitates microelectrodes that can be singularly addressed and arranged in a high-density topography. This thesis provides two fabrication solutions that comply with these requirements and allow to manufacture highly conductive vertical platinum microelectrodes with high aspect-ratio. According to the two processes reported, the three-dimensional (3D) cores of the electrodes are fabricated in SU-8 or in silicon respectively. These tridimensional structures are successively coated by a metal layer, after a passivation step in the case of silicon. The planar metal connections which singularly address the free-standing microelectrodes are patterned differently for the two approaches, respectively by lift-off and spray coating. Importantly, the 3D microelectrodes can be co-fabricated with microfluidic structures to obtain multiple active sites for single-cell analysis. In this thesis, in the framework of a collaboration with Ludwig Centre for Cancer Biology (Lausanne, Switzerland), the microelectrodes have been employed to detect activated T cells. The encouraging results pave the way to a new generation of microfluidic platform based on 3D microelectrodes to attain real-time and label-free monitoring of individual T cells to employ in immunotherapy

Miniaturized bioanalytics to probe the function of membrane proteins

G-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped

Advanced dielectrophoretic cell separation systems

This thesis describes experimental and theoretical investigations into new particle handling and separation methods and techniques. It makes a major contribution to the rapidly expanding field of cell separation technology. A novel dielectrophoretic cell separation system has been developed, which is capable of processing large sample volumes (~50mL) in a flow through system. Previously reported dielectrophoretic cell separator systems typically process sample volumes in the 100mL range. The electrode configuration developed for this work allows the isolation and concentration of single particle types from large sample volumes; a method which could be further developed into a new rare-cell separation technology. In addition, a new technique of particle fractionation was developed termed ‘Dielectrophoretic Chromatography’. A cell separation chip was designed and built using standard micro-fabrication techniques. Experimental work was undertaken to demonstrate the function and limitations of the device. Numerical modelling of the particle motion in the device is presented and compared with experimental work for a number of different particle types, applied voltages and fluid flow rates. The dielectrophoretic separation system comprises a microfluidic channel, of cross-section 100mm x 10mm and length 50mm, with two sets of interdigitated microelectrode arrays. The first set of arrays, with characteristic electrode size 40mm, called a focussing device, has electrodes patterned onto the top and bottom surfaces of the flow channel. The second electrode array, which is part of the same device, has an electrode array patterned only on the bottom of the channel. Two sizes of secondary electrode array were used 20mm and 40mm. AC voltages (from 1V to 10V peak) are applied to the microelectrode, with a frequency between 10kHz to 180MHz. A dielectrophoretic force is exerted on the particles as they flow along the channel. The first electrode array uses negative dielectrophoresis to focus the stream of particles entering the device into a narrow sheet (one particle diameter thick) midway between the upper and lower channel surfaces. The second electrode array, down stream from the first is separately controllable

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

Tools for single cell proteomics

Despite recent advances that offer control of single cells, in terms of manipulation and sorting and the ability to measure gene expression, the need to measure protein copy number remains unmet. Measuring protein copy number in single cells and related quantities such as levels of phosphorylation and protein-protein interaction is the basis of single cell proteomics. A technology platform to undertake the analysis of protein copy number from single cells has been developed. The approach described is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by mechanical shearing caused by laser-induced microcavitation; and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. This demonstrates the ability count protein copy number from single cells in a manner which could be applied in principle to any set of proteins and for any cell type without the need for genetic engineering. Metabolism can undergo alteration in diseases such as cancer and heart failure and also as cells differentiate during development. In order to assess how it may inform a proteomic measurement, multidimensional two-photon fluorescence metabolic imaging is conducted on a cultured cancer cell line, primary adult rat cardiomyocytes and human embryonic stem cells. By measuring the parameters of fluorescence such as intensity and lifetime of the autofluorescent metabolic co-factors NADH and FAD, it was found to be possible to contrast cells under various conditions and metabolic stimuli. In particular, human embryonic stem cells were able to be contrasted at 3 stages of development as they underwent differentiation into embryonic stem cell derived cardiomyocytes. Metabolic imaging provides a non-destructive method to monitor cellular metabolic activity with high resolution. This is complimentary to the single cell proteomic platform and the convergence of both techniques holds promise in future investigations into how metabolism influences cell function and the proteome in development and disease