An impedance based single cell deformability cytometer

Abstract

The mechanical characteristics of biological cells are determined by the properties of the cytosol, membrane, organelles and the cytoskeleton, a network of filaments that extend throughout the cell. Mechanical interactions between cells and the extracellular matrix (ECM) are linked to proliferation of cancerous cells and to leukocytes activation occurring during infections. Mechanical properties of single biological cells can be used to distinguish cancerous cells from healthy ones and can indicate the presence of infections and their measurement does not need any fluorescence tag. Label-free methods have the advantage of fewer preparation steps, reduced use of reagents and limited invasivity on the sample, which can be reused for further analysis. To evaluate mechanical properties, deformability is measured by monitoring the change of shape under a given applied stress. Several methods, such as micropipette aspiration, atomic force microscopy, magnetic twisting and optical traps have been developed. However, they can analyse only up to ~1 cell per minute. Such a low throughput is inadequate to analyse large samples and identify rare events. High-throughput in-flow deformability cytometers have been recently developed, exploiting hydrodynamic forces to deform the cells. However, the use of optical systems to evaluate cell shape limits the potential of these solutions for point-of- care applications. This work describes a high-throughput cytometer which evaluates the deformability of single cells under hydrodynamic induced stress by measuring their impedance along different axes using microelectrodes placed on the top and the bottom of a microchannel. The device can distinguish cells treated with glutaraldehyde, which makes their membrane stiffer. The ratio of the parameter defined as electrical circularity between treated and untreated cells was estimated in 0.62±0.05, corresponding to an optical circularity of 0.975±0.013 measured using a custom dark-field microscope attached to the chip, used as a verification method. Further studies on the effect of Cytochalasin D, a cytoskeletal drug that affects the polymerisation of actin filaments, and TGFβ1, a protein involved in cells differentiation, show interesting, yet not conclusive results, with the optical measurements apparently contradicting the impedance measurements. It is possible that biophysical, in addition to geometrical aspects may play an important role, with the opening of mechano-sensitive ion channels and transient pores on the lipid membrane potentially contributing to the overall impedance. One of the underlying causes of the global threat of antibiotic resistance is the lack of rapid methods of pathogen identification and antibiotic susceptibility tests. In this work an optimised Microfluidic Impedance Cytometer (MIC) chip has been manufactured with a limit of detection of 0.75 μm in diameter, capable to detect and size bacteria (1.58±0.26 μm for E. Coli and 1.04±0.16 μm for Ps. Aeruginosa) and distinguish them also based on their dialectical properties when stimulated at around 7.5 MHz. Although this technique can detect bacteria in a purified medium, their identification in a clinical sample is challenging, due to the large amount of debris typically found in an infected sample. It was also demonstrated, although just in a preliminary test, that this technique can be used as a rapid antibiotic susceptibility test, with ofloxacine reducing the bacterial growth rate of Pseudomonas Aeruginosa in such a way that the total count after only 3 hours incubation was reduced by ~1/4 at a concentration of 50 μg/ml and by ~1/5 at 200 μg/ml with respect to a nontreated sample.

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