Advances in Microfluidic Impedance Cytometry for Electromechanical Characterization of Cells

Abstract

Single-cell analysis is extremely important for discovering unique characteristics of individual cells, identifying cell populations of interest, and understanding their behaviour during disease conditions. With the advent of flow cytometry - a well-established technique for counting, identifying and sorting cells, single-cell studies have progressed rapidly over the past few decades. Conventional flow cytometers are generally expensive pieces of machinery and utilize complex fluorescent detection systems for the analysis of single cells. Impedance cytometers, in contrast, offer label-free sample preparation, reduced experimental costs, lower sample volumes, simpler operation, and better portability. In the recent past, impedance cytometers have been used to analyse the electrical properties of numerous cell types in a rapid and efficient manner. However, its usage in understanding cell mechanical properties is sparsely explored. Early attempts in this aspect have been accompanied by major limitations in terms of device configurations, electrode geometries, and throughput. This thesis, thus, aims to overcome these limitations by exploring novel techniques for the electromechanical analysis of single-cells.

To begin with, we propose a novel microfluidic impedance sensing platform capable of independently and simultaneously characterizing the electrical and mechanical properties of cells. Using healthy and chemically-stiffened erythrocytes, we validate the platform and discuss how it overcomes several challenges associated with traditional impedance cytometers. We then improve upon this platform by introducing a simple yet innovative flow-correction technique to circumvent flow-rate fluctuations in syringe-pump driven fluid flow, thereby accelerating data acquisition and improving accuracy. Using this upgraded platform, we offer valuable insight into the differences in electromechanical properties of human lymphocytes in healthy and diabetic individuals.

The next part of this thesis deals with the discovery of a unique frequency-dependent phenomenon in impedance cytometers that are characterized by the presence of “double peaks” in the reactive component of the impedance response from single cells. Through extensive simulations, we initially probed the origins of this effect and how various cell parameters affect its behaviour. In particular, the phenomenon was found to be highly sensitive to changes in cell membrane capacitance and it was subsequently exploited to demonstrate a novel experimental technique for distinguishing cell types based on variations in the dielectric properties of their cell membrane. This technique allowed the measurement of both cell size and cell dielectric properties at a single low frequency (400-800 kHz) rather than using multiple frequencies through opacity measurements (impedance at high frequency/impedance at low frequency) as has been done till date in conventional impedance cytometry.

Building upon the previous study, we demonstrate how the double peak effect can be used to electrically measure the deformability of cells, a task that traditionally involves expensive high-speed cameras and time-consuming image processing. Through an extensive FEM study, we identify optimum electrode geometries and probing frequencies for analysing the double peak phenomenon in the case of squeezed and deformed cells in microfluidic constrictions. We show how sub-micron changes in cell deformation can be precisely measured through simple peak detection schemes.

The final part of this thesis discusses the design and development of an FPGA-based lock-in amplifier for the electrical detection of cells. Compared to conventional digital lock-in amplifiers, FPGA-based systems permit parallel processing and are easily programmable, which enhances its versatility. We showcase a proof-of-concept platform by integrating an impedance flow cytometer with the FPGA-based lock-in amplifier to capture and analyse individual electrical signals from cells. Using the phase-sensitive measurement principle, we discuss how the system detects the cell signal at a particular frequency and rejects the rest. We further comment on noise reduction capabilities and throughput. This platform is expected to significantly reduce instrumentation costs and pave the way for the development of cheaper, portable, and more versatile impedance flow cytometers.