Single-cell impedance analysis and sorting

Abstract

Microfluidic impedance cytometry (MIC) is a label-free technique for differentiating cell phenotypes according to their inherent biophysical markers, principally mechanical (deformability) and dielectric (membrane capacitance and cytoplasm conductivity) properties. This thesis describes new approaches for the measurement of both parameters coupled with a novel method for real-time sorting of single cells according to their electro-mechanical phenotype.
Single-cell microfluidic impedance spectroscopy has been limited in frequency range and in the ability to extract the full set of electrical parameters from single cells. In this thesis, the measurement frequency range was extended to 500MHz, enabling full characterisation of single nucleated cells at high throughput. System validation used HL60 cells and THP-1 cells (differentiated into macrophages) exposed to different chemical treatments in order to change their electrical properties. It was shown that suspending the cells in saline of lower conductivity enhanced discrimination between the cell types and treatment. Single cell spectra were fitted to the double-shell model to obtain membrane capacitance and cytoplasm conductivity.
Different single-cell deformability cytometers have been developed, but these are mostly based on processing high-speed optical images of single cells undergoing deformation. This thesis describes a prototype microfluidic deformability cytometer that measures both the electrical and optical deformability. It simultaneously measures the optical and electrical shape change of single cells deformed in a viscoelastic shear flow. Optical deformability is measured using a low-cost CMOS camera with images of single cells generated with a short LED pulse triggered by an impedance signal from a cell. Electrical deformability of the cell is determined by electrode arrays that measure the shape changes along two axes. The system was characterised by measuring the deformability of HL60 cells treated with cytoskeleton disrupting chemicals. Results show an excellent correlation between the optical and electrical methods.
Finally, a single-cell sorting system was developed by integrating membrane pumps into the micro-cytometer. When the membranes are displaced, they generate short pressure pulses that can deflect single cells into different outlets. An algorithm was developed to generate a trigger according to the particle velocity. Proof of principle experiments demonstrate successful sorting of cells and beads based on size with a throughput of 13 particles/s and purity of 97%.

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Identifiers

ORCID for Xueping Zou: orcid.org/0009-0000-7946-565X

ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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