The past decade has seen substantial growth in research into how changes in the biomechanical and biophysical properties of cells and subcellular structures influence, and are influenced by, the onset and progression of human diseases. This paper presents an overview of the rapidly expanding, nascent field of research that deals with the biomechanics and biophysics of cancer cells. The review begins with some key observations on the biology of cancer cells and on the role of actin microfilaments, intermediate filaments and microtubule biopolymer cytoskeletal components in influencing cell mechanics, locomotion, differentiation and neoplastic transformation. In order to set the scene for mechanistic discussions of the connections among alterations to subcellular structures, attendant changes in cell deformability, cytoadherence, migration, invasion and tumor metastasis, a survey is presented of the various quantitative mechanical and physical assays to extract the elastic and viscoelastic deformability of cancer cells. Results available in the literature on cell mechanics for different types of cancer are then reviewed. Representative case studies are presented next to illustrate how chemically induced cytoskeletal changes, biomechanical responses and signals from the intracellular regions act in concert with the chemomechanical environment of the extracellular matrix and the molecular tumorigenic signaling pathways to effect malignant transformations. Results are presented to illustrate how changes to cytoskeletal architecture induced by cancer drugs and chemotherapy regimens can significantly influence cell mechanics and disease state. It is reasoned through experimental evidence that greater understanding of the mechanics of cancer cell deformability and its interactions with the extracellular physical, chemical and biological environments offers enormous potential for significant new developments in disease diagnostics, prophylactics, therapeutics and drug efficacy assays.

New studies suggest that stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications.

The traditional analytical biosensor instruments are relatively bulky, expensive, and not easy to handle, thus their applications are largely limited in resource-limited settings. The recent development of microfluidic lab-on-a-chip (LOC) technology has provided a possible solution to miniaturize the conventional biosensing system, yet other accessory devices to detect, readout, analyze, transfer, and display results are still required. With the rapid development, mass production, and pervasive distribution of smartphones in recent years, they have provided people with portable, cost-effective, and easy-to-operate platforms to build analytical biosensors for point-of-care (POC) applications and mobile health. Based on the common analytical methods, this paper reviews the recent development of four types of smartphone based analytical biosensory systems at the POC, i.e., smartphone-based microscopic imaging, colorimetric, electrochemical, and electrochemiluminescence biosensor. The different bio-sensing strategies and analytical performance together with future perspectives are discussed.

Biophysical (mechanical and electrical) properties of living cells have been proven to play important roles in the regulation of various biological activities at the molecular and cellular level, and can serve as promising label-free markers of cells' physiological states. In the past two decades, a number of research tools have been developed for understanding the association between the biophysical property changes of biological cells and human diseases; however, technical challenges of realizing high-throughput, robust and easy-to-perform measurements on single-cell biophysical properties have yet to be solved. In this paper, we review emerging tools enabled by microfluidic technologies for single-cell biophysical characterization. Different techniques are compared. The technical details, advantages, and limitations of various microfluidic devices are discussed.

We propose a model to determine the influence of different cell properties, such as size, membrane capacitance and cytoplasm conductivity, on the impedance spectrum as measured in a microfabricated cytometer. A dielectric sphere of equivalent complex permittivity is used as a simplified model to describe a biological cell. The measurement takes place between a pair of facing microelectrodes in a microchannel filled with a saline solution. The model incorporates various cell parameters, such as dielectric properties, size and position in the channel. A 3D finite element model is used to evaluate the magnitude of the electric field in the channel and the resultant changes in charge densities at the measurement electrode boundaries as a cell flows past. The charge density is integrated on the electrode surface to determine the displacement current and the channel impedance for the computed frequency range. The complete impedance model combines the finite element model, the electrode-electrolyte interface impedance and stray impedance, which are measured from a real device. The modeled dielectric complex spectra for various cell parameters are discussed and a measurement strategy for cell discrimination with such a system is proposed. We finally discuss the amount of noise and measurement fluctuations of the sensor.

A new cytological tool, based on the microCoulter particle counter (microCPC) principle, aimed at diagnostic applications for cell counting and separation in haematology, oncology or toxicology is described. The device measures the spectral impedance of individual cells or particles and allows screening rates over 100 samples s(-1) on a single-cell basis. This analyzer is intended to drive a sorting actuator producing a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput. 3D finite element simulations are presented to compare various electrode geometries and their influence on cell parameters estimation. The device is based on a glass-polyimide microfluidic chip with integrated channels and electrodes microfabricated at the length scale of the particles to be investigated (1-20 microm). A laminar liquid flow carries the suspended particles through the measurement area. Each particle's impedance signal is recorded by a differential pair of microelectrodes using the cell surrounding media as a reference. The micromachined chip and processing electronic circuit allow simultaneous impedance measurements at multiple frequencies, ranging from 100 kHz to 15 MHz. In this paper, we describe the microfabrication and characterisation of an on-chip flow-cytometer as the first building block of a complete cell-sorting device. We then discuss the signal conditioning technique and finally impedance measurements of cells and particles of different sizes and types to demonstrate the differentiation of subpopulations in a mixed sample.

This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.This journal is © The Royal Society of Chemistry 2011

Mechanical properties of cells, reflective of various biochemical characteristics such as gene expression and cytoskeleton, are promising label-free biomarkers for studying and characterizing cells. Electrical properties of cells, dependent on the cellular structure and content, are also label-free indicators of cell states and phenotypes. In this work, we have developed a microfluidic device that is able to simultaneously characterize the mechanical and electrical properties of individual biological cells in a high-throughput manner (>1000 cells/min). The deformability of MCF-7 breast cancer cells was characterized based on the passage time required for an individual cell to pass through a constriction smaller than the cell size. The total passage time can be divided into two components: the entry time required for a cell to deform and enter a constriction, which is dominated by the deformability of cells, and the transit time required for the fully deformed cell to travel inside the constriction, which mainly relies on the surface friction between cells and the channel wall. The two time durations for individual cells to pass through the entry region and transit region have both been investigated. In addition, undeformed cells and fully deformed cells were simultaneously characterized via electrical impedance spectroscopy technique. The combination of mechanical and electrical properties serves as a unique set of intrinsic cellular biomarkers for single-cell analysis, providing better differentiation of cellular phenotypes, which are not easily discernible via single-marker analysis.

A wavy-herringbone (wavy-HB) structured microfluidic device was used to effectively and selectively capture and release circulating tumor cells (CTCs) by using immunoaffinity and magnetic force. This device was designed to create passive turbulence and increase the possibility of tumor cells colliding with the device wall. Under an external magnetic field, magnetic particles (MPs) coated with anti-EpCAM against a tumor cell surface protein (EpCAM) were immobilized over the wavy-HB surface to capture tumor cells. After removing the magnetic field, the captured cells with surplus MPs were released from the device and collected; thus, these cells could be re-cultured for further analysis. Under optimized conditions, the capture efficiency of the tumor cells can be as high as 92% ± 2.8%. Capture experiments were also performed on whole blood samples, and the capture efficiency was in a high range of 81-95%, at different tumor cell concentrations. Such a method can potentially be used for CTC sorting from patient blood samples, CTC concentration monitoring, therapeutic guidance and drug dosage choice, and further study of tumors, such as drug screening and tumor mutations.

Motion analysis of optically trapped objects is demonstrated using a simple 2D Fourier transform technique. The displacements of trapped objects are determined directly from the phase shift between the Fourier transform of subsequent images. Using end- and side-view imaging, the stiffness of the trap is determined in three dimensions. The Fourier transform method is simple to implement and applicable in cases where the trapped object changes shape or where the lighting conditions change. This is illustrated by tracking a fluorescent particle and a myoblast cell, with subsequent determination of diffusion coefficients and the trapping forces.

Single-cell impedance measurement is a label-free, noninvasive method for characterizing the electrical properties of single cells. At present, though widely used for impedance measurement, electric impedance flow cytometry (IFC) and electric impedance spectroscopy (EIS) are used alone for most microfluidic chips. In this paper, we present a microfluidic device combining the IFC and EIS techniques for single-cell electrical property measurement. The device uses hydrodynamic constriction to passively trap single cells and uses coplanar electrodes to obtain the impedance spectrum of the trapped cell via EIS and discrete impedance data points of the passing cells via IFC. Through experiment, we verified the individual functionality of IFC and EIS respectively, by revealing through IFC the impedance magnitude difference and quantifying through EIS the area-specific membrane capacitance and cytoplasm conductivity of the three types of cancer cells. We also demonstrated the complementarity of IFC and EIS, which holds for a wide range of the flow rate. We envision that the strategy of combining IFC and EIS provides a new thought in the efforts to enhancing the efficiency of electrical property measurement for single cells.

High-resolution microscopic imaging may cause intensive image processing and potential impact of light irradiation on yeast replicative lifespan (RLS). Electrical impedance spectroscopy (EIS) could be alternatively used to perform high-throughput and label-free yeast RLS assays. Prior to fabricating EIS-integrated microfluidic devices for yeast RLS determination, systematic modeling and theoretical investigation are crucial for device design and optimization. Here, we report three-dimensional (3D) finite-element modeling and simulations of EIS measurement in a microfluidic single yeast in-situ impedance array (SYIIA), which is designed by patterning an electrode matrix underneath a cell-trapping array. SYIIA was instantiated and modeled as a 5×5 sensing array comprising 25 units for cell immobilization, culturing and time-lapse EIS recording. Simulations of yeast growing and budding in a sensing unit demonstrated that EIS signals enable the characterization of cell growth and daughter-cell dissections. In the 5×5 sensing array, simulation results indicated that when monitoring a target cell, daughter dissections in its surrounding traps may induce variations of the recorded EIS signals, which could cause mistakes in identifying target daughter-cell dissections. To eliminate the mis-identifications, electrode array pitch was optimized. Therefore, the results could conduct the design and optimization of microfluidic electrode-array-integrated devices for high-throughput and accurate yeast RLS assays. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

Microfluidic impedance cytometry offers a simple non-invasive method for single-cell analysis. Coplanar electrode chips are especially attractive due to ease of fabrication, yielding miniaturized, reproducible, and ultimately low-cost devices. However, their accuracy is challenged by the dependence of the measured signal on particle trajectory within the interrogation volume, that manifests itself as an error in the estimated particle size, unless any kind of focusing system is used. In this paper, we present an original five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted. This metric is exploited to correct the estimated size of polystyrene beads of 5.2, 6 and 7 μm nominal diameter, reaching coefficient of variations lower than the manufacturers' quoted values. The potential impact of the proposed device in the field of life sciences is demonstrated with an application to Saccharomyces cerevisiae yeast.

Submicron-precision particle characterization is crucial for counting, sizing and identifying a variety of biological particles, such as bacteria and apoptotic bodies. Microfluidic impedance cytometry has been attractive in current research for microparticle characterization due to its advantages of label-free detection, ease of miniaturization and affordability. However, conventional electrode configurations of three electrodes and floating electrodes have not yet demonstrated the capability of probing submicron particles or microparticles with a submicron size difference. In this study, we present a label-free high-throughput (∼800 particles per second) impedance-based microfluidic flow cytometry system integrated with a novel design of a double differential electrode configuration, enabling submicron particle detection (down to 0.4 μm) with a minimum size resolution of 200 nm. The signal-to-noise ratio has been boosted from 13.98 dB to 32.64 dB compared to a typical three-electrode configuration. With the proposed microfluidic impedance cytometry, we have shown results of sizing microparticles that accurately correlate with manufacturers' datasheets (R2 = 0.99938). It also shows that population ratios of differently sized beads in mixture samples are consistent with the results given by commercial fluorescence-based flow cytometry (within ∼1% difference). This work provides a label-free approach with submicron precision for sizing and counting microscale and submicron particles, and a new avenue of designing electrode configurations with a feature of suppressing the electrical noise for accomplishing a high signal-to-noise ratio in a wide range of frequencies. This novel double differential impedance sensing system paves a new pathway for real-time analysis and accurate particle screening in pathological and pharmacological research.

We present an innovative impedance cytometer for the measurement of the cross-sectional position of single particles or cells flowing in a microchannel. As predicted by numerical simulations and experimentally validated, the proposed approach is applicable to particles/cells with either spherical or non-spherical shape. In particular, the optics-free high-throughput position detection of individual flowing red blood cells (RBCs) is demonstrated and applied to monitor RBCs hydrodynamic focusing under different sheath flow conditions. Moreover, the device provides multiparametric information useful for lab-on-a-chip applications, including particle inter-arrival times and velocity profile, as well as RBCs mean corpuscular volume, distribution width and electrical opacity.

An impedance spectroscopy-based cell/particle position detection method in microfluidic systems is presented. A single pair of non-parallel surface microelectrodes was utilized to detect the transverse positions of particles/cells flowing in a microchannel without the need for a multi-electrode multi-channel impedance detection. This method can be a simple solution for high-throughput and low-cost position detection in microfluidic sorting and separation applications.

This paper describes a new design of microfluidic impedance cytometer enabling accurate characterization of particles without the need for focusing. The approach uses multiple pairs of electrodes to measure the transit time of particles through the device in two simultaneous different current measurements, a transverse (top to bottom) current and an oblique current. This gives a new metric that can be used to estimate the vertical position of the particle trajectory through the microchannel. This parameter effectively compensates for the non-uniform electric field in the channel that is an unavoidable consequence of the use of planar parallel facing electrodes. The new technique is explained and validated using numerical modelling. Impedance data for 5, 6 and 7 μm particles are collected and compared with simulations. The method gives excellent coefficient of variation in (electrical) radius of particles of 1% for a sheathless configuration.

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 10 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.

单细胞水平的检测能够在细胞群中分辨出稀有的异常细胞,在生物医学领域如疾病的早期诊断和治疗评估等方面有着至关重要的作用。通过整合微流控技术、电阻抗技术与流式细胞术,微流控阻抗细胞仪能够在微流体精确操控条件下,实现流动态单细胞的连续、无损阻抗检测。与传统的单细胞检测方法相比,微流控阻抗细胞仪具有非标记、多参数、低污染和检测速度快等显著优势,为细胞的种类鉴别与状态监测提供了强有力的工具,因此近年来研究学者已成功开发出各式具有不同结构和功能的微流控阻抗细胞仪。本文首先介绍直流阻抗细胞仪、交流阻抗细胞仪以及形变阻抗细胞仪的工作原理和开发进展,随后讨论微流控阻抗细胞仪在血细胞、癌细胞和微生物等生物样品检测中的最新应用情况,并从集成化和微型化两方面阐述微流控阻抗细胞仪在临床即时检测中的应用前景,最后总结了现有微流控阻抗细胞仪存在的不足并探讨了该领域的未来发展趋势。

Biophysical markers of cells such as cellular electrical and mechanical properties have been proven as promising label-free biomarkers for studying, characterizing, and classifying different cell types and even their subpopulations. Further analysis or manipulation of specific cell types or subtypes requires accurate isolation of them from the original heterogeneous samples. However, there is currently a lack of cell sorting ability that could actively separate a large number of individual cells at the single-cell level based on their multivariate biophysical makers or phenotypes. In this work, we, for the first time, demonstrate label-free and high-throughput acoustic single-cell sorting activated by the characterization of multivariate biophysical phenotypes. Electrical phenotyping is implemented by single-cell electrical impedance characterization with two pairs of differential sensing electrodes, while mechanical phenotyping is performed by extracting the transit time for the single cell to pass through microconstriction from the recorded impedance signals. A real-time impedance signal processing and triggering algorithm has been developed to identify the target sample population and activate a pulsed highly focused surface acoustic wave for single-cell level sorting. We have demonstrated acoustic single-particle sorting solely based on electrical or mechanical phenotyping. Furthermore, we have applied the developed microfluidic system to sort live MCF-7 cells from a mixture of fixed and live MCF-7 population activated by a combined electrical and mechanical phenotyping at a high throughput >100 cells/s and purity ∼91.8%. This demonstrated ability to analyze and sort cells based on multivariate biophysical phenotyping provides a solution to the current challenges of cell purification that lack specific molecular biomarkers.

This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electrical impedance spectroscopy in order to perform a theoretical study to clarify our results. This study focuses on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy.

Microtissue spheroids in microfluidic devices are increasingly used to establish novel in vitro organ models of the human body. As the spheroids are comparably sizable, it is difficult to monitor larger numbers of them by optical means. Therefore, electrical impedance spectroscopy (EIS) emerges as a viable alternative to probing spheroid properties. Current spheroid EIS systems are, however, not suitable for investigating multiple spheroids in parallel over extended time in an automated fashion. Here we address this issue by presenting an automated, multiplexed EIS (AMEIS) platform for impedance analysis in a microfluidic setting. The system was used to continuously monitor the effect of the anticancer drug fluorouracil (5-FU) on HCT116 cancer spheroids. Simultaneous EIS monitoring of up to 15 spheroids was performed in parallel over 4 days at a temporal resolution of 2 min without any need for pumps. The measurements were continuous in nature, and the setup was kept in a standard incubator under controlled conditions during the measurements. A baseline normalization method to improve robustness and to reduce the influence of slow changes in the medium conductivity on the spheroid EIS readings has been developed and validated by experiments and means of a finite-element model. The same method and platform was then used for online monitoring of cardiac spheroids. The beating frequency of each cardiac spheroid could be read out in a completely automated fashion. The developed system constitutes a promising method for simultaneously evaluating drug impact and/or toxic effects on multiple microtissue spheroids.

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 µm wide and 30 µm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 µm and 2 µm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.