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

DEVELOPMENT OF HIGH-THROUGHPUT IMPEDANCE SPECTROSCOPY-BASED MICROFLUIDIC PLATFORM FOR DETECTING AND ANALYZING CELLS AND PARTICLES

Impedance spectroscopy based microfluidics have the capability to characterize the dielectric properties of mediums, particles, cellular and sub-cellular contents in response to stimulating voltage signals over a frequency range. This label-free technology has broad ranges of applications in life sciences where there is a need for high-throughput, label-free, non-contact, and low-cost microsystems. To address these limitations, three innovative impedance spectroscopy microfluidic platforms have been developed and presented in this dissertation. The first platform was developed for detecting and characterizing the transverse position of a single cell flowing within a microfluidic channel using a single impedance spectroscopy electrode pair. Regardless of the cell separation methods used, identifying and quantifying the position of cells and particles within a microchannel are important, as these information indicate both the degree of separation as well as how many cells are separated into each position. Using a single pair of non-parallel surface microelectrodes, five different transverse positions of single cells flowing through a microfluidic channel were successfully identified at a throughput of more than 400 particles/s using the detected impedance peak height and width. The second platform utilizes the above technology to count and quantify cells flowing through multiple outlets of microfluidic cell separation systems. A single pair of step-shaped electrodes was developed by integrating five different electrode-to-electrode gaps within a single pair of electrodes. Using this platform, an overall misclassification error rate of only 1.85% was achieved. The result shows the technology’s capability in achieving efficient on-chip cell counting and quantification, regardless of the cell separation methods used, making it a promising on-chip, low-cost and label-free quantification method for cell and particle sorting and separation applications. The third platform was developed for counting cells and particles encapsulated in water-in-oil emulsion droplets using microfluidic based impedance spectroscopy systems. Impedance signal peak height and width were utilized to successfully quantify the number of cells encapsulated within a droplet, and was successfully applied for various cell types and growth media. In addition, the developed platform has been also successfully tested for identifying and discriminating filamentous fungal cell growth, where single fungal spores and filamentous fungi of different lengths could be discriminated inside droplets. Overall in this research, several impedance spectroscopy based microfluidic systems have been successfully developed to solve current limitations in technologies that need high-throughput, low-cost and label-free detection and characterization method for a broad range of cell/particle screening applications

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

Test analysis & fault simulation of microfluidic systems

This work presents a design, simulation and test methodology for microfluidic systems, with particular focus on simulation for test. A Microfluidic Fault Simulator (MFS) has been created based around COMSOL which allows a fault-free system model to undergo fault injection and provide test measurements. A post MFS test analysis procedure is also described.A range of fault-free system simulations have been cross-validated to experimental work to gauge the accuracy of the fundamental simulation approach prior to further investigation and development of the simulation and test procedure.A generic mechanism, termed a fault block, has been developed to provide fault injection and a method of describing a low abstraction behavioural fault model within the system. This technique has allowed the creation of a fault library containing a range of different microfluidic fault conditions. Each of the fault models has been cross-validated to experimental conditions or published results to determine their accuracy.Two test methods, namely, impedance spectroscopy and Levich electro-chemical sensors have been investigated as general methods of microfluidic test, each of which has been shown to be sensitive to a multitude of fault. Each method has successfully been implemented within the simulation environment and each cross-validated by first-hand experimentation or published work.A test analysis procedure based around the Neyman-Pearson criterion has been developed to allow a probabilistic metric for each test applied for a given fault condition, providing a quantitive assessment of each test. These metrics are used to analyse the sensitivity of each test method, useful when determining which tests to employ in the final system. Furthermore, these probabilistic metrics may be combined to provide a fault coverage metric for the complete system.The complete MFS method has been applied to two system cases studies; a hydrodynamic “Y” channel and a flow cytometry system for prognosing head and neck cancer.Decision trees are trained based on the test measurement data and fault conditions as a means of classifying the systems fault condition state. The classification rules created by the decision trees may be displayed graphically or as a set of rules which can be loaded into test instrumentation. During the course of this research a high voltage power supply instrument has been developed to aid electro-osmotic experimentation and an impedance spectrometer to provide embedded test

Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry

Multiparameter cell-tracking intrinsic cytometry for single-cell characterization

An abundance of label-free microfluidic techniques for measuring cell intrinsic markers exists, yet these techniques are seldom combined because of integration complexity such as restricted physical space and incompatible modes of operation. We introduce a multiparameter intrinsic cytometry approach for the characterization of single cells that combines ≥2 label-free measurement techniques onto the same platform and uses cell tracking to associate the measured properties to cells. Our proof-of-concept implementation can measure up to five intrinsic properties including size, deformability, and polarizability at three frequencies. Each measurement module along with the integrated platform were validated and evaluated in the context of chemically induced changes in the actin cytoskeleton of cells. viSNE and machine learning classification were used to determine the orthogonality between and the contribution of the measured intrinsic markers for cell classification

Raman-spectroscopy based cell identification on a microhole array chip

Circulating tumor cells (CTCs) from blood of cancer patients are valuable prognostic markers and enable monitoring responses to therapy. The extremely low number of CTCs makes their isolation and characterization a major technological challenge. For label-free cell identification a novel combination of Raman spectroscopy with a microhole array platform is described that is expected to support high-throughput and multiplex analyses. Raman spectra were registered from regularly arranged cells on the chip with low background noise from the silicon nitride chip membrane. A classification model was trained to distinguish leukocytes from myeloblasts (OCI-AML3) and breast cancer cells (MCF-7 and BT-20). The model was validated by Raman spectra of a mixed cell population. The high spectral quality, low destructivity and high classification accuracy suggests that this approach is promising for Raman activated cell sorting