Impedance Cytometers: Label-Free Counting of Enriched Cells.

Impedance measurements of biological cells is a label-free, noninvasive, and quantitative analytical method. Impedance sensing is based on measuring the impedance from a frequency-dependent voltage signal (i.e., excitation signal) applied to the target. Impedance measurements provide the opportunity for high-throughput analysis of cells and particles, which is convenient when a large number of particles or cells need to be characterized and counted. Solsona et al.¹⁵⁴ addressed challenges related to the detection of the exact position of cells or particles as they enter the sensing zone between the electrodes when impedance data are collected. Using platinum electrodes fabricated as an array with a gradually increasing surface area, the authors demonstrated that an electric field gradient in a parallel two electrode array allowed for tracking of particle position in one axis. By using low frequency measurements, it was possible to detect the exact position of the particles. At higher frequencies, where the impedance of the medium dominates and the current density is homogeneous throughout the entire electrochemical cell, the conductivity of particles or cells can be measured. Studies used a finite element model to define the frequency range at which both the position and conductivity of the particles or cells can be detected and measured. The model was validated with microparticles using a microfluidic chip with linearly increasing electrodeposited areas perpendicular to the flow direction.

Petchakup et al.¹⁵⁵ reported on leukocytes sorted from blood using a Dean flow fractionation device and follow-up impedance profiling of sorted cells (Figure 9A,​,B).B). The detector consisted of a three-electrode setup; the middle electrode was supplied with potential at two different frequencies and the differential current response from cells was recorded from the side electrodes and sent to a lock-in amplifier to extract a magnitude and phase of the signal of interest. In this application, a microfluidic impedance flow cytometer was used for testing and characterizing neutrophils and monocytes obtained from patients with type 2 diabetes. Here, leukocyte dielectric properties were associated with cardiovascular risk factors. Additionally, tested cells demonstrated pro-inflammatory phenotypes, which would imply that leukocyte impedance signatures could be used as a biomarker of inflammation. The method was also able to monitor monocyte differentiation and provide biophysical characterization of monocyte subsets.

(A) Dean flow fractionation device: (a) workflow for leukocyte impedance phenotyping for sample preprocessing, DFF leukocyte sorting, and impedance profiling, (b) image of the microfluidic chips, (c) optical image of single cells flowing through the electrodes in the detection region, and (d) measurement of multiple events from single cells. Reprinted from Biosens. Bioelectron., Vol. 118, Petchakup, C.; Tay, H. M.; Yeap, W. H.; Dalan, R.; Wong, S. C.; Li, K. H. H.; Hou, H. W. Label-free leukocyte sorting and impedance-based profiling for diabetes testing, pp 195–203 (ref ¹⁵⁵). Copyright 2018, with permission from Elsevier. (B) Impedance profiling of different blood cell samples: (a) density scatter plot of cell size (|Z_LF|, V) versus opacity of diluted whole blood, PBMCs, DFF-sorted monocytes, DFF-sorted lymphocytes, and DFF-sorted neutrophils and (b) frequency distribution of different leukocyte subtypes. Reprinted from Biosens. Bioelectron., Vol. 118, Petchakup, C.; Tay, H. M.; Yeap, W. H.; Dalan, R.; Wong, S. C.; Li, K. H. H.; Hou, H. W. Label-free leukocyte sorting and impedance-based profiling for diabetes testing, pp 195–203 (ref ¹⁵⁵). Copyright 2018, with permission from Elsevier. (C) Sensing area showing a simple analytical expression for the lateral position measurement of the flowing particles (derived from the electrical signal, positions of the flowing particles, electrodes, and microchannel). Reproduced from Yang, D.; Ai, Y. Lab Chip 2019, 19, 3609–3617 (ref ¹⁵⁶), with permission of The Royal Society of Chemistry. (D) Schematic of a microfluidic impedance cytometer showing impedance and fluorescence detection sections. The fluorescence from cells was measured simultaneously with impedance allowing direct correlation of electrical and fluorescent properties of single cells. Reproduced from Honrado, C.; Ciuffreda, L.; Spencer, D.; Ranford-Cartwright, L.; Morgan, H. J. R. Soc. Interface 2018, 15, 20180416 (ref ³³), with permission of The Royal Society of Chemistry.

Yang et al.,¹⁵⁶ similar to the work of Solsona et al.,¹⁵⁴ demonstrated an impedance sensor consisting of a three electrode detector with an N-shaped design. The microfluidic impedance flow cytometer was designed in that way to provide information on the lateral position of measured cells and particles. A differential current collected from the N-shaped electrodes encoded the trajectory of flowing single particles (Figure 9C).

Research results presented by Honrado et al.³³ evaluated changes in biophysical properties of RBCs during the malaria infection cycle using an impedance sensor (Figure 9D). Microfluidic impedance cytometry measured the dielectric properties of Plasmodium falciparum-infected RBCs, and the results demonstrated that the membrane capacitance and cytoplasmic conductivity of infected RBCs increased during the time course of the infection, possibly correlating with the increasing volume occupied by the parasite. The authors suggested that their findings could be used for the development of DEP-based sorting of RBCs as a standard parasite detection protocol during post ring-stage of the infection cycle with increasing sensitivity over current diagnostic methods. The cell or particle lateral position was derived from the measured electrical signal and distance relationship between the positions of particles, electrodes, and microchannels. The results demonstrated that the device could measure the lateral position of single particles and cells while simultaneously characterizing their size.