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An impedance-coupled microfluidic device for single-cell analysis of primary cell wall regeneration
Abstract
Primary cell wall (PCW) is a rigid yet flexible cell wall surrounding plant cells and it plays key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. As a technique widely used to study the characteristics of mammalian cells, electrical impedance spectroscopy (EIS) is rarely used in plant science. In this work, we designed and fabricated an EIS based biosensor coupled with microfluidic platform to investigate the formation process of primary cell wall (PCW) at the single-cell level. Arabidopsis mesophyll cells with completely regenerated PCW showed significantly higher impedance values compared to the nascent protoplasts without PCW, demonstrating that PCW formation caused a dramatic change in cell electrical properties. The device could also discriminate plant mutant cells with modified PCW compositions, thus provided a novel tool for physical phenotyping of plant cells. The dose-dependent effects of exogenously applied auxin on PCW regeneration were corroborated on this platform which revealed its potential to sensitively detect the influences of in vitro stimuli. This work not only provided one novel application of impedance-based biosensor to characterize a plant-specific developmental event, but also revealed the promises of EIS integrated microfluidic system as a sensitive, time-effective and low-cost platform to characterize single plant cells and make new scientific discoveries in plant science.
... Han et al. developed an IFC device to characterize the biophysical properties of two model plant species, herbaceous Arabidopsis thaliana and woody Populus trichocarpa [60]. In the regeneration process of primary cell wall (PCW), plant cells are gradually covered by the fibrillary network, which becomes thick and interlaced, resulting in the decrease of capacitance of cell membrane and PCW [148]. Thus, the researchers found that the Arabidopsis cells with regenerated PCW were less deformable and electrically conductive than that without PCW. ...
... Recent EIS sensing devices applied in single-cell analysis are summarized in Table 2. These devices are classified into two categories: one is to determine the optimal frequency at which the impedance of different cell lines or cell states is most sensitive [82,150] and the other is to continuously monitor the dynamic cell process or cell behavior and phenotypic changes [83,85,91,92,94,95,148,[151][152][153]. EIS sensing technology has been used to investigate the optimal frequency at which the characteristic parameters extracted from EIS signals are most prominent in measuring specific dielectric properties of cells [82,150]. ...
... (E) Imaginary part of current response for Arabidopsis mesophyll cells at different status (0 h, 12 h and 24 h after incubation, respectively). Reproduced from [148] with the permission from Elsevier. (F) The Bode impedance spectra measured on working electrode before and after cell migration, as well as on reference electrode without cells over the frequency range from 100 Hz to 1 MHz. ...
Cellular heterogeneity is of significance in cell-based assays for life science, biomedicine and clinical diagnostics. Electrical impedance sensing technology has become a powerful tool, allowing for rapid, non-invasive, and label-free acquisition of electrical parameters of single cells. These electrical parameters, i.e., equivalent cell resistance, membrane capacitance and cytoplasm conductivity, are closely related to cellular biophysical properties and dynamic activities, such as size, morphology, membrane intactness, growth state, and proliferation. This review summarizes basic principles, analytical models and design concepts of single-cell impedance sensing devices, including impedance flow cytometry (IFC) to detect flow-through single cells and electrical impedance spectroscopy (EIS) to monitor immobilized single cells. Then, recent advances of both electrical impedance sensing systems applied in cell recognition, cell counting, viability detection, phenotypic assay, cell screening, and other cell detection are presented. Finally, prospects of impedance sensing technology in single-cell analysis are discussed.
... The measured and extracted signal depends not only on the dielectric microstructures of the cell but also on the particle trajectory within the measuring area for characterization of the dielectric properties of particles and cells as they pass through a microchannel with integrated electrodes [20], thus challenging the resolution and accuracy of the technique. So, the main target of the previous studies is to improve the cell focusing techniques to avoid the effect of the cell [21] position on the extracted signal to enhance the accuracy and the resolution of the extracted signal. For example, in the hydrodynamic focusing system [21,22], The technique focuses cells on a fluid solution within the optical system's focal point using a sheath fluid medium. ...
... So, the main target of the previous studies is to improve the cell focusing techniques to avoid the effect of the cell [21] position on the extracted signal to enhance the accuracy and the resolution of the extracted signal. For example, in the hydrodynamic focusing system [21,22], The technique focuses cells on a fluid solution within the optical system's focal point using a sheath fluid medium. The sheath fluid medium's purpose is to keep contaminants and clumped clusters from obstructing the channel. ...
The Dielectrophoresis force delivers an effective and appropriate way to control particles, especially for cell-selective manipulation and cell separation. It has been applied in cell sorting and drug development. Dielectrophoresis force has been applied in the cell trapping process, cell alignment, separation, and isolation of different sized particles, which are applied in a variety of biological applications. Micro electric impedance spectroscopy (µEIS) are very tiny devices that use fluid as a working medium in conjunction with biological cells to extract different electrical parameters. Many advantages can be provided by using these tiny microfluidic devices, such as portability, disposable, and high accuracy.
Polystyrene beads are extensively utilized as reference particles to show the efficiency and to validate quantitatively the detection of Dielectrophoresis systems. In this work, two different forms of trapping dielectrophoresis force combined with a new strategy of impedance extraction are presented and discussed. The study aims to enhance the microfluidic system to differentiate between cells and particles with low conductivity and different sizes. The analysis technique is combined with impedance spectroscopy in a single microfluidic chip that not only enables efficient trapping of cells but also enhances the electric impedance of cells in a label-free and non-invasive using different sizes of Polystyrene Particles.
... It is important to note that these amplifiers may have different bandwidths; thus, it depends on the sample to choose the best lock-in amplifier for a particular experiment. Various studies have discussed the selection of frequencies for specific samples such as bacteria, 20 plants, 75 and mammalian cells. 66 ...
Imaging and impedance flow cytometry is a label-free technique that has shown promise as a potential replacement for standard flow cytometry. This is due to its ability to provide rich information and archive high-throughput analysis. Recently, significant efforts have been made to leverage machine learning for processing the abundant data generated by those techniques, enabling rapid and accurate analysis. Harnessing the power of machine learning, imaging and impedance flow cytometry has demonstrated its capability to address various complex phenotyping scenarios. Herein, we present a comprehensive overview of the detailed strategies for implementing machine learning in imaging and impedance flow cytometry. We initiate the discussion by outlining the commonly employed setup to acquire the data (i.e., image or signal) from the cell. Subsequently, we delve into the necessary processes for extracting features from the acquired image or signal data. Finally, we discuss how these features can be utilized for cell phenotyping through the application of machine learning algorithms. Furthermore, we discuss the existing challenges and provide insights for future perspectives of intelligent imaging and impedance flow cytometry.
... This technology has many advantages such as: Ability to manipulate and detect small volumes, Low consumption of reagents, Less human error, Higher repeatability, Quick system response and Reduction of power consumption [2]. Recently, this technology has been used in a wide range of applications such as biological systems [3], Lab on-chip systems [4,5], microelectromechanical devices (MEMS) [6], microreactors [7] and micromixers [8]. In microfluidics, the system used for the mixing process in small length scales is called the micromixer. ...
Micromixers are one of the common pieces of equipment in microfluidic devices and have been widely used for a variety of applications. They are generally divided into active and passive categories. Active micromixers use external energy sources for mixing. Pressure-based micromixers are a common type of active micromixer that use a variety of strategies to create disturbances in the flow and improve mixing. Of all the strategies used for this type of micromixer, oscillating bubble injection has rarely been investigated. In this study, we investigate the flow characteristics and mass transfer process in a two-dimensional micromixer based on oscillating bubble injection. Different parameters affect the mixing performance in this micromixer. In this study, the effects of Reynolds number, bubble oscillation frequency, bubble oscillation amplitude, and side channel geometry on three variables of mixing index, pressure drop, and air inlet pressure have been investigated. The results show that with increasing Reynolds number at constant bubble oscillation frequency and amplitude, the mixing index decreases. Also, with increasing bubble oscillation frequency at constant Reynolds number and constant oscillation amplitude, the mixing index will increase. Increasing the bubble oscillation amplitude at constant Reynolds number and constant oscillation frequency will increase the mixing index. In a simple side channel with a constant diameter, the bubble becomes unstable, so this geometry is undesirable. Changing the side channel geometry by introducing an expansion in the side channel will solve this problem. Nevertheless, increasing the side channel expansion width will reduce the mixing index. The best mixing index was 78.19% in 4.5 seconds, and it shows that the proposed micromixer of this research has good performance in a relatively short time.
... Thus, defining the parameters, such as applied voltage and frequency, are essential for analysis to improve the system's reliability in extracting cell dielectric features [21,27,28], including cancer, stem, and neural cells [25]. µEIS has been used to study the properties of tissues and organelles, including changes in their composition or structure [29]. Researchers [30] also performed an impedance analysis on human cervical cancer cell lines as a function of frequency. ...
Background
Microelectrical Impedance Spectroscopy (µEIS) is a tiny device that utilizes fluid as a working medium in combination with biological cells to extract various electrical parameters. Dielectric parameters of biological cells are essential parameters that can be extracted using µEIS. µEIS has many advantages, such as portability, disposable sensors, and high-precision results.
Results
The paper compares different configurations of interdigitated microelectrodes with and without a passivation layer on the cell contact tracks. The influence of the number of electrodes on the enhancement of the extracted impedance for different types of cells was provided and discussed. Different types of cells are experimentally tested, such as viable and non-viable MCF7, along with different buffer solutions. This study confirms the importance of µEIS for in vivo and in vitro applications. An essential application of µEIS is to differentiate between the cells’ sizes based on the measured capacitance, which is indirectly related to the cells’ size. The extracted statistical values reveal the capability and sensitivity of the system to distinguish between two clusters of cells based on viability and size.
Conclusion
A completely portable and easy-to-use system, including different sensor configurations, was designed, fabricated, and experimentally tested. The system was used to extract the dielectric parameters of the Microbeads and MCF7 cells immersed in different buffer solutions. The high sensitivity of the readout circuit, which enables it to extract the difference between the viable and non-viable cells, was provided and discussed. The proposed system can extract and differentiate between different types of cells based on cells’ sizes; two other polystyrene microbeads with different sizes are tested. Contamination that may happen was avoided using a Microfluidic chamber. The study shows a good match between the experiment and simulation results. The study also shows the optimum number of interdigitated electrodes that can be used to extract the variation in the dielectric parameters of the cells without leakage current or parasitic capacitance.
... Plant physiological states are reflected by changes in their ion content, membrane permeability, and viscosity. Electrical impedance spectroscopy can be used to rapidly and nondestructively examine these changes; however, this method is constrained by the plant barrier layer [137]. The combination of microneedles and an impedance measurement sensor overcomes this restriction, as microneedles can pass through the cuticle (Fig. 9C) [138]. ...
Similar to blood, interstitial fluid (ISF) contains exogenous drugs and biomarkers and may therefore substitute blood in drug analysis. However, current ISF extraction techniques require bulky instruments and are both time-consuming and complicated, which has inspired the development of viable alternatives such as those relying on skin or tissue puncturing with microneedles. Currently, microneedles are widely employed for transdermal drug delivery and have been successfully used for ISF extraction by different mechanisms to facilitate subsequent analysis. The integration of microneedles with sensors enables in situ ISF analysis and specific compound monitoring, while the integration of monitoring and delivery functions in wearable devices allows real-time dose modification. Herein, we review the progress in drug analysis based on microneedle-assisted ISF extraction and discuss the related future opportunities and challenges.
... In higher plants, the primary cell wall (PCW) is a highly organized structure consisting of crystalline cellulose microfibrils embedded in a hydrated matrix of pectin and hemicellulose [9]. The primary cell wall was stripped during protoplast production and will regenerate over the next 24 to 48 h [46]. Next, we investigated the physiological role of auxin under normal or UV-B conditions in the process of primary cell wall regeneration. ...
A better understanding of the phenotypic heterogeneity of protoplasts requires a comprehensive analysis of the morphological and metabolic characteristics of many individual cells. In this study, we developed a microfluidic flow cytometry with fluorescence sensor for functional characterization and phenotyping of protoplasts to allow an unbiased assessment of the influence of environmental factors at the single cell level. First, based on the measurement of intracellular homeostasis of reactive oxygen species (ROS) with a DCFH-DA dye, the effects of various external stress factors such as H2O2, temperature, ultraviolet (UV) light, and cadmium ions on intracellular ROS accumulation in Arabidopsis mesophyll protoplasts were quantitatively investigated. Second, a faster and stronger oxidative burst was observed in Petunia protoplasts isolated from white petals than in those isolated from purple petals, demonstrating the photoprotective role of anthocyanins. Third, using mutants with different endogenous auxin, we demonstrated the beneficial effect of auxin during the process of primary cell wall regeneration. Moreover, UV-B irradiation has a similar accelerating effect by increasing the intracellular auxin level, as shown by double fluorescence channels. In summary, our work has revealed previously underappreciated phenotypic variability within a protoplast population and demonstrated the advantages of a microfluidic flow cytometry for assessing the in vivo dynamics of plant metabolic and physiological indices at the single-cell level.
... The measurement frequency was changed incrementally from 10kHz, 50kHz, 100kHz, 500kHz, 800kHz, 1MHz and 2 MHz, and the applied constant voltage was set at 1 V. The selected frequencies from 100 kHz to 2 MHz were used in this study, to concentrate on the impedance variations caused by the membrane lipid and to minimize the effect of electrical double-layer capacitance [56]. With the same procedure for microbead detection, the impedance of single RBCs was measured and collected. ...
Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution, and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially-available Tungsten needle coated with parylene. When a single cell flows through the parallel facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m ⁻² , with the regression coefficients, E at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.
... Current endpoint analysis techniques like histology and fluorescent microscopy rely on fixing chemicals and stains that modify or destroy the cells, preventing continuous, real-time analysis. A non-destructive assay like EIS does not have this effect and has been successfully applied to in-vitro monitoring of cell wall regeneration and antineoplastic drug assessment [26] [27]. ...
In this work, a novel microfluidic cell culture platform capable of automated electrical impedance measurements and immunofluorescence and brightfield microscopy was developed for further in-vitro cellular research intended to optimize cell culture conditions. The microfluidic system design, fabrication, automation, and design verification testing are described. Electrical and optical measurements of the 16 parallel cell culture chambers were automated via a custom LabView interface. A proposed design change will enable gas diffusion, removing the need for an environmental enclosure and allow long-term cell culture experiments. This "lab on a chip" system miniaturizes and automates experiments improving testing throughput and accuracy while creating a highly controllable microenvironment for cell culture. Such a system can be applied to drug development, bioassays, diagnostics, and animal testing alternatives. This work is part of a collaborative effort to define protocols for the electrical and optical characterization of cell culture within a novel microfluidic device with the intent of optimizing microenvironment conditions.
... Hahn et al., 2020;Guyon-Debast et al., 2021;Nicolia et al., 2021). Another example is the adaptation of efficient and low-cost microfluidic techniques to perform spatiotemporal studies of plant protoplasts physiology during their development (Sakai et al., 2019) and to apprehend the electrical resistance of CW-regenerated protoplasts (Chen, 2020). Similarly, the usefulness of protoplasts for high-throughput RNA sequencing has also been put forward due to its many advantages over traditional RNA-seq. ...
Plants are constantly facing abiotic and biotic stresses. To continue to thrive in their environment, they have developed many sophisticated mechanisms to perceive these stresses and provide an appropriate response. There are many ways to study these stress signals in plant, and among them, protoplasts appear to provide a unique experimental system. As plant cells devoid of cell wall, protoplasts allow observations at the individual cell level. They also offer a prime access to the plasma membrane and an original view on the inside of the cell. In this regard, protoplasts are particularly useful to address essential biological questions regarding stress response, such as protein signaling, ion fluxes, ROS production, and plasma membrane dynamics. Here, the tools associated with protoplasts to comprehend plant stress signaling are overviewed and their potential to decipher plant defense mechanisms is discussed.
... Small sample volume, high surface to volume ratio, short processing time, and high throughput procedures are among these advantages [2][3][4][5][6][7][8]. These devices have been widely used in chemistry and biomedical applications, including drug delivery [9][10][11], cell separation and manipulation [12-15[16] [56]], single-cell analysis [17][18][19][20][21][22], etc. Particle separations have many applications in biomedical and chemical studies. For example, cell separation is applicable in therapeutics, cell biology, and diagnostics [23]. ...
Microfluidics devices are widely used for particle separation. Deterministic Lateral Displacement (DLD) is one of the passive methods for particle separation. DLD devices mainly separate particles based on their sizes. There are two main modes of movement in DLD arrays; the small particles move in a zigzag path, and the larger particles separate in the displacement mode. So, it is important to estimate the critical particle size for the transition of modes before the fabrication of DLD devices. Asymmetry in the design of the arrays can affect the fluid behavior and the critical particle size. In this numerical study, we investigated the effects of the asymmetry caused by changing the downstream gap size to the lateral gap size ratio on the fluid behavior and particle trajectories in DLD devices. We used two dimensional (2D) Finite Element Method (FEM) to study the variations in the flow lane's widths and combined the fluid analysis with structural mechanics to model the contact between the particles and the posts in DLD arrays. We simulated the spherical particles' trajectories with diameters ranging from to 19.2 in circular post DLD arrays with a lateral gap size of. In contrast to the previous works, in these simulations, the effect of particle movement on the fluid flow profiles was considered. We evaluated the particle movement mode in seven different values of the downstream gap size to the lateral gap size ratio (rang-ing from 0.5 to 2) and eight different row shift fraction (ranging from 0.025 to 0.3). Our simulations showed that increasing the value of the downstream gap while the lateral gap is fixed increases the veering flow rate and width. By finding the particle with the largest diameter in the zigzag mode and the particle with the smallest diameter in the displacement mode, we estimated the critical particle diameter for each value of shift fraction in different values of the downstream gap to the lateral gap size ratio. Using these data, a curve was fitted for predicting the critical particle diameter in each ratio. Finally, a more general form of the formula for the critical particle diameter was proposed, which considers an extra parameter compared to the previous ones. The results of this study can lead to a better understanding of DLD devices' functions and, thus, save time and costs for better designs and experiments.
... However, continuous EIS measurement was not accessible in such system, since the impedance signals were recorded instantaneously when cells were flowing through the detection region. By incorporating single-cell trapping structures into microfluidic devices, in-situ EIS offers the capacity to monitor cellular dynamic processes of cells during a longtime period, such as migration, spread and death of HeLa cell [37], cell differentiation [38], and cell wall regeneration [39]. Moreover, we have patterned coplanar microelectrodes underneath bottleneck-like single-cell traps to successfully measure the cell growth of budding yeast [40], and monitor the cell cycle of fission yeast, Schizosaccharomyces pombe (S. pombe) over an extended time period [41]. ...
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
... Recently, EIS was extended to single plant cells for studying primary cell wall regeneration. 115 Raman Spectroscopy. Surface-enhanced Raman scattering (SERS) is attractive due to its low limit of detection and high spatial resolution. ...
Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m-2, with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.
As a label-free single cell analysis approach, impedance flow cytometry provides valuable information by differential signals. However, for high throughput purpose, particle coincidence is inevitable especially when the cell concentration or pumping rate is too high. In this work, we proposed the first numerical model and a novel method for particle coincidence. To investigate the multi-particle events, a double differential impedance sensor is used. From a statistic analysis of signal peaks, we could determine the threshold of particle events, and then read out particle numbers. Compared with customized threshold, the data-driven cut-off line fundamentally improves the accuracy of particle recognition. Herein, the key operation parameters that influence the particle coincidence including sample concentration, driven pressure (stands for pumping rate), and sensor configuration are elucidated. Moreover, a guide to the best practices for avoid multi-particle coincidence events are suggested for real applications.
Impedance cytometry is a well-established technique for counting and analyzing single cells, with several advantages, such as convenience, high throughput, and no labeling required. A typical experiment consists of the following steps: single-cell measurement, signal processing, data calibration, and particle subtype identification. At the beginning of this article, we compared commercial and self-developed options extensively and provided references for developing reliable detection systems, which are necessary for cell measurement. Then, a number of typical impedance metrics and their relationships to biophysical properties of cells were analyzed with respect to the impedance signal analysis. Given the rapid advances of intelligent impedance cytometry in the past decade, this article also discussed the development of representative machine learning-based approaches and systems, and their applications in data calibration and particle identification. Finally, the remaining challenges facing the field were summarized, and potential future directions for each step of impedance detection were discussed.
Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.
A core challenge in biomedical and life science research is to decipher cell-to-cell differences under pathological progression or external perturbation. Microfluidics-based integrated systems serve as powerful tools for accurately investigating cellular phenotypes and molecular responses in the state of physiological and pathological processes at the single-cell scale. Here, we review the advanced microfluidic strategies for single-cell research relevant to tumorigenesis and evolution, immune responses, and drug discovery. The review will discuss the design of microfluidic devices for single-cell analysis, the current methods of microfluidic devices for single-cell isolation, and applications of microfluidics-based single-cell analysis in cell biology, disease diagnosis, and therapy. Specifically, we will focus on the microfluidics-based techniques for single-cell operation and recent strategies for single-cell measurements. Limitations and future perspectives of microfluidics-based single-cell analysis will also be discussed.
Single-cell impedance analysis can provide valuable information for characterizing and discriminating cells. In this paper, a cost-effective portable microfluidic impedance cytometer (MIC) was proposed to realize the broadband impedance analysis of cells by using maximum length sequence (MLS) and viscoelastic focusing. The MIC comprised a microfluidic chip with a straight microchannel, two indium tin oxide (ITO) electrodes, and a home-made platform performing maximum length sequence technique. The viscoelastic focusing enabled cells to focus into a single train to eliminate the influence of cell position variation on acquired electrical signals and allow the cells to pass through the detection region one by one. The MLS technique realized the fast broadband impedance detection of single cells at a low hardware cost. The impedance data under multiplex frequencies was obtained to uncover the dielectric properties of white blood cells (WBCs) and MCF-7 cancer cells. The machine learning was used to train the impedance data and to identify cell types. The results indicated that 98.98% of MCF-7 cells and 98.65% of WBCs were correctly identified. Our MIC showed a potential to be developed as a cost-effective and portable device for point-of-care testing of circulating tumor cells from patients’ peripheral blood.
At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.
Despite that single-cell-type-level analyses have been extensively conducted on animal models to gain new insight into complex biological processes, the unique biological and physiological properties of plant cells have not been widely studied at single-cell resolution. In this work, an electrical impedance flow cytometry was fabricated based on microfluidics with constriction microchannel to simultaneously characterize the mechanical and electrical properties of single plant cells. Protoplasts from two model plant species, the herbaceous Arabidopsis thaliana and the woody Populus trichocarpa, could be readily discriminated by their respective mechanical traits, but not by electrical impedance. On the contrary, overexpression of a red fluorescent protein (RFP) on plasma membrane resulted in changes in cell electrical impedance instead of cell deformability. During primary cell wall (PCW) regeneration, this extracellular layer outside of protoplasts introduced dramatic variations in both mechanical and electrical properties of single plant cells. Furthermore, the effects of auxin, an essential phytohormone regulating PCW reformation, were validated on this platform. Taken together, our results revealed a novel application of microfluidic impedance flow cytometry in the field of plant science to simultaneously characterize dual biophysical properties at single cell resolution, which could be further developed as a powerful and reliable tool for plant cell phenotyping and cell fate specification.
To address the need for the on-site measurement of aging oil, in this paper, we propose an impedance-based microsensor for analyzing the moisture content in engine oil. Using a microfabrication process, we fabricated an interdigitated microelectrode and integrated it with a 3D-printed microcontainer to produce a microsensor that can detect changes in the permittivity of oil. When the moisture content in oil increases, this sensor can detect the resulting change in the oil impedance, which is related to its permittivity, and then determine the degree to which the oil has aged. The test results show that the proposed microsensor has the advantages of being small and having high sensitivity, good accuracy, and the ability to be combined with hand-held instruments. The proposed method is expected to be used for the rapid, low cost, on-site determination of oil aging.
Background:
Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Methods and differentiation protocols used in plant physiology and biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes.
Results:
Herein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens (P. patens) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin.
Conclusions:
We developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods.
Plant cells are surrounded by highly dynamic cell walls that play important roles regulating aspects of plant development. Recent advances in visualization and measurement of cell wall properties have enabled accumulation of new data about wall architecture and biomechanics. This has resulted in greater understanding of the dynamics of cell wall deposition and remodeling. The cell wall is the first line of defense against different adverse abiotic and biotic environmental influences. Different abiotic stress conditions such as salinity, drought, and frost trigger production of Reactive Oxygen Species (ROS) which act as important signaling molecules in stress activated cellular responses. Detection of ROS by still-elusive receptors triggers numerous signaling events that result in production of different protective compounds or even cell death, but most notably in stress-induced cell wall remodeling. This is mediated by different plant hormones, of which the most studied are jasmonic acid and brassinosteroids. In this review we highlight key factors involved in sensing, signal transduction, and response(s) to abiotic stress and how these mechanisms are related to cell wall-associated stress acclimatization. ROS, plant hormones, cell wall remodeling enzymes and different wall mechanosensors act coordinately during abiotic stress, resulting in abiotic stress wall acclimatization, enabling plants to survive adverse environmental conditions.
The bulk of a plant's biomass, termed secondary cell walls, accumulates in woody xylem tissues and is largely recalcitrant to biochemical degradation and saccharification1. By contrast, primary cell walls, which are chemically distinct, flexible and generally unlignified2, are easier to deconstruct. Thus, engineering certain primary wall characteristics into xylem secondary walls would be interesting to readily exploit biomass for industrial processing. Here, we demonstrated that by expressing AP2/ERF transcription factors from group IIId and IIIe in xylem fibre cells of mutants lacking secondary walls, we could generate plants with thickened cell wall characteristics of primary cell walls in the place of secondary cell walls. These unique, newly formed walls displayed physicochemical and ultrastructural features consistent with primary walls and had gene expression profiles illustrative of primary wall synthesis. These data indicate that the group IIId and IIIe AP2/ERFs are transcription factors regulating primary cell wall deposition and could form the foundation for exchanging one cell wall type for another in plants.
Plant cell walls provide structural support for growth and serve as a barrier for pathogen attack. Plant cell walls are also a source of renewable biomass for conversion to biofuels and bioproducts. Understanding plant cell wall biosynthesis and its regulation is of critical importance for the genetic modification of plant feedstocks for cost-effective biofuels and bioproducts conversion and production. Great progress has been made in identifying enzymes involved in plant cell wall biosynthesis, and in Arabidopsis it is generally recognized that the regulation of genes encoding these enzymes is under a transcriptional regulatory network with coherent feedforward and feedback loops. However, less is known about the transcriptional regulation of plant secondary cell wall biosynthesis in woody species despite of its high relevance to biofuels and bioproducts conversion and production. In this article, we synthesize recent progress on the transcriptional regulation of secondary cell wall biosynthesis in Arabidopsis and contrast to what is known in woody species. Furthermore, we evaluate progress in related emerging regulatory machineries targeting transcription factors in this complex regulatory network of secondary cell wall biosynthesis.
Cellulose consists of linear chains of β-1,4-linked glucose units, which are synthesized by the cellulose synthase complex (CSC). In plants, these chains associate in an ordered manner to form the cellulose microfibrils. Both the CSC and the local environment in which the individual chains coalesce to form the cellulose microfibril determine the structure and the unique physical properties of the microfibril. There are several recent reviews that cover many aspects of cellulose biosynthesis, which include trafficking of the complex to the plasma membrane and the relationship between the movement of the CSC and the underlying cortical microtubules (Bringmann et al. 2012 Trends Plant Sci. 17 , 666–674 ( doi:10.1016/j.tplants.2012.06.003 ); Kumar & Turner 2015 Phytochemistry 112 , 91–99 ( doi:10.1016/j.phytochem.2014.07.009 ); Schneider et al. 2016 Curr. Opin. Plant Biol. 34 , 9–16 ( doi:10.1016/j.pbi.2016.07.007 )). In this review, we will focus on recent advances in cellulose biosynthesis in plants, with an emphasis on our current understanding of the structure of individual catalytic subunits together with the local membrane environment where cellulose synthesis occurs. We will attempt to relate this information to our current knowledge of the structure of the cellulose microfibril and propose a model in which variations in the structure of the CSC have important implications for the structure of the cellulose microfibril produced.
This article is part of a discussion meeting issue ‘New horizons for cellulose nanotechnology’.
Vibrational spectroscopy provides non-destructively the molecular fingerprint of plant cells in the native state. In combination with microscopy, the chemical composition can be followed in context with the microstructure, and due to the non-destructive application, in-situ studies of changes during, e.g., degradation or mechanical load are possible. The two complementary vibrational microspectroscopic approaches, Fourier-Transform Infrared (FT-IR) Microspectroscopy and Confocal Raman spectroscopy, are based on different physical principles and the resulting different drawbacks and advantages in plant applications are reviewed. Examples for FT-IR and Raman microscopy applications on plant cell walls, including imaging as well as in-situ studies, are shown to have high potential to get a deeper understanding of structure–function relationships as well as biological processes and technical treatments. Both probe numerous different molecular vibrations of all components at once and thus result in spectra with many overlapping bands, a challenge for assignment and interpretation. With the help of multivariate unmixing methods (e.g., vertex components analysis), the most pure components can be revealed and their distribution mapped, even tiny layers and structures (250 nm). Instrumental as well as data analysis progresses make both microspectroscopic methods more and more promising tools in plant cell wall research.
At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.
The plant cell wall has a diversity of functions. It provides a structural framework to support plant growth and acts as the first line of defense when the plant encounters pathogens. The cell wall must also retain some flexibility, such that when subjected to developmental, biotic, or abiotic stimuli it can be rapidly remodeled in response. Genes encoding enzymes capable of synthesizing or hydrolyzing components of the plant cell wall show differential expression when subjected to different stresses, suggesting they may facilitate stress tolerance through changes in cell wall composition. In this review we summarize recent genetic and transcriptomic data from the literature supporting a role for specific cell wall-related genes in stress responses, in both dicot and monocot systems. These studies highlight that the molecular signatures of cell wall modification are often complex and dynamic, with multiple genes appearing to respond to a given stimulus. Despite this, comparisons between publically available datasets indicate that in many instances cell wall-related genes respond similarly to different pathogens and abiotic stresses, even across the monocot-dicot boundary. We propose that the emerging picture of cell wall remodeling during stress is one that utilizes a common toolkit of cell wall-related genes, multiple modifications to cell wall structure, and a defined set of stress-responsive transcription factors that regulate them.
Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population.
Impedance measurement of live biological cells is widely accepted as a label free, non-invasive and quantitative analytical method to assess cell status. This method is easy-to-use and flexible for device design and fabrication. In this review, three typical techniques for impedance measurement, i.e., electric cell-substrate impedance sensing, Impedance flow cytometry and electric impedance spectroscopy, are reviewed from the aspects of theory, to electrode design and fabrication, and applications. Benefiting from the integration of microelectronic and microfluidic techniques, impedance sensing methods have expanded their applications to nearly all aspects of biology, including living cell counting and analysis, cell biology research, cancer research, drug screening, and food and environmental safety monitoring. The integration with other techniques, the fabrication of devices for certain biological assays, and the development of point-of-need diagnosis devices is predicted to be future trend for impedance sensing techniques.
CESA5 synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis thaliana. The involvement of additional CESA proteins in this process and details concerning the manner in which the cellulose is deposited in the mucilage pocket are unknown. Here we show that both CESA3 and CESA10 are also highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant 1 (ixr1-1 and ixr1-2) mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and GFP-tagged CESA5, CESA3, and CESA10 proteins moved in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion as well as mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.
Copyright © 2015, Plant Physiology.
This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.
Plants exposed to abiotic stress respond to the unfavorable conditions at multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all of the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs.
Electrical impedance spectroscopy (EIS) is a noninvasive method for characterizing the dielectric properties of biological particles. The technique can differentiate between cell types and provide information on cell properties through measurement of the permittivity and conductivity of the cell membrane and cytoplasm. In terms of lab-on-a-chip (LOC) technology, cells pass sequentially through the microfluidic channel at high speed and are analyzed individually, rather than as traditionally done on a mixture of particles in suspension. This paper describes the analytical and numerical modeling methods for EIS of single cell analysis in a microfluidic cytometer. The presented modeling methods include Maxwell's mixture theory, equivalent circuit model and finite element method. The difference and advantages of these methods have been discussed. The modeling work has covered the static case — an immobilized cell in suspension and the dynamic case — a moving cell in the channel.
We report on the development of a vertical and transparent microfluidic chip for high-throughput phenotyping of Arabidopsis thaliana plants. Multiple Arabidopsis seeds can be germinated and grown hydroponically over more than two weeks in the chip, thus enabling large-scale and quantitative monitoring of plant phenotypes. The novel vertical arrangement of this microfluidic device not only allows for normal gravitropic growth of the plants but also, more importantly, makes it convenient to continuously monitor phenotypic changes in plants at the whole organismal level, including seed germination and root and shoot growth (hypocotyls, cotyledons, and leaves), as well as at the cellular level. We also developed a hydrodynamic trapping method to automatically place single seeds into seed holding sites of the device and to avoid potential damage to seeds that might occur during manual loading. We demonstrated general utility of this microfluidic device by showing clear visible phenotypes of the immutans mutant of Arabidopsis, and we also showed changes occurring during plant-pathogen interactions at different developmental stages. Arabidopsis plants grown in the device maintained normal morphological and physiological behaviour, and distinct phenotypic variations consistent with a priori data were observed via high-resolution images taken in real time. Moreover, the timeline for different developmental stages for plants grown in this device was highly comparable to growth using a conventional agar plate method. This prototype plant chip technology is expected to lead to the establishment of a powerful experimental and cost-effective framework for high-throughput and precise plant phenotyping.
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.
The plant cell wall mostly comprises complex glycans, which are synthesized by numerous enzymes located in the Golgi apparatus and plasma membrane. Protein-protein interactions have been shown to constitute an important organizing principle for glycan biosynthetic enzymes in mammals and yeast. Recent genetic and biochemical data also indicate that such interactions could be common in plant cell wall biosynthesis. In this review, we examine the new findings in protein-protein interactions among plant cell wall biosynthetic enzymes and discuss the possibilities for enzyme complexes in the Golgi apparatus. These new insights in the field may contribute to novel strategies for molecular engineering of the cell wall.
Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.
The plant hormone auxin, in particular indole-3-acetic acid (IAA), is a key regulator of virtually every aspect of plant growth and development. Auxin regulates transcription by rapidly modulating levels of Aux/IAA proteins throughout development. Recent studies demonstrate that auxin perception occurs through a novel mechanism. Auxin binds to TIR1, the F-box subunit of the ubiquitin ligase complex SCF(TIR1), and stabilizes the interaction between TIR1 and Aux/IAA substrates. This interaction results in Aux/IAA ubiquitination and subsequent degradation. Regulation of the Aux/IAA protein family by TIR1 and TIR1-like auxin receptors (AFBs) links auxin action to transcriptional regulation and provides a model by which the vast array of auxin influences on development may be understood. Moreover, auxin receptor function is the first example of small-molecule regulation of an SCF ubiquitin ligase and may have important implications for studies of regulated protein degradation in other species, including animals.
A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively).
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The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.
Electrochemical biosensors combine the sensitivity of electroanalytical methods with the inherent bioselectivity of the biological component. The biological component in the sensor recognizes its analyte resulting in a catalytic or binding event that ultimately produces an electrical signal monitored by a transducer that is proportional to analyte concentration. Some of these sensor devices have reached the commercial stage and are routinely used in clinical, environmental, industrial, and agricultural applications. The two classes of electrochemical biosensors, biocatalytic devices and affinity sensors, will be discussed in this critical review to provide an accessible introduction to electrochemical biosensors for any scientist (110 references).
The design, fabrication techniques and characterization of an on-chip flow- cytometer device as well as the packaging approach is described. The device consists of planar microelectrodes situated on one or both sides of a micro- channel connected to an electronic detection circuitry. Simulations of the cells effect on the channel impedance are presented and compared with real measurements. Efforts have particularly been focused on realizing an interconnnecting setup to provide easy electrical and fluidic connections, to allow good monitoring and easy handling of the chip. Our cell analyzer is aimed at diagnostic applications for cell counting and separation in hematology (blood cells, leukocytes and reticulocytes). Another possible application is a micro-cell sorter that can be used to separate abnormal cell types that are present in samples at very low number such as cancer cells (cancer cytology).
Mammalian fibroblasts have been cultured on evaporated gold electrodes subjected to an alternating electric field at 4000 Hz. The system consists of a large (approximately equal to 2 cm2) and a small (approximately equal to 3 X 10(-4) cm2) electrode bathed in tissue culture medium. The applied electric field produces a voltage drop at the boundary between the solution and the small electrode of a few mV at a current density of a few mA/cm2. The small population of cells that attach and spread on this electrode have a marked effect on the measured impedance and also cause it to fluctuate with time. The amplitude of these fluctuations is greatly reduced by cytochalasin B (10 microM), suggesting they are a consequence of cell movement.
Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.
Cellulose, an abundant, crystalline polysaccharide, is central to plant morphogenesis and to many industries. Chemical and ultrastructural analyses together with map-based cloning indicate that the RSW1 locus of Arabidopsis encodes the catalytic subunit of cellulose synthase. The cloned gene complements the rsw1 mutant whose temperature-sensitive allele is changed in one amino acid. The mutant allele causes a specific reduction in cellulose synthesis, accumulation of noncrystalline beta-1,4-glucan, disassembly of cellulose synthase, and widespread morphological abnormalities. Microfibril crystallization may require proper assembly of the RSW1 gene product into synthase complexes whereas glucan biosynthesis per se does not.
Analysis of food, pharmaceutical, and environmental compounds is an inevitable issue to evaluate quality of the compounds used in human life. Quality of drinking water, food products, and pharmaceutical compounds is directly associated with human health. Presence of forbidden additives in food products, toxic compounds in water samples and drugs with low quality lead to important problems for human health. Therefore, attention to analytical strategy for investigation of quality of food, pharmaceutical, and environmental compounds and monitoring presence of forbidden compounds in materials used by humans has increased in recent years. Analytical methods help to identify and quantify both permissible and unauthorized compounds present in the materials used in human daily life. Among analytical methods, electrochemical methods have been shown to have more advantages compared to other analytical methods due to their portability and low cost. Most of big companies have applied this type of analytical methods because of their fast and selective analysis. Due to simple operation and high diversity of electroanalytical sensors, these types of sensors are expected to be the future generation of analytical systems. Therefore, many scientists and researchers have focused on designing and fabrication of electroanalytical sensors with good selectivity and high sensitivity for different types of compounds such as drugs, food, and environmental pollutants. In this paper, we described the mechanism and different examples of DNA, enzymatic and electro-catalytic methods for electroanalytical determination of drug, food and environmental compounds.
The architecture of the plant cell wall is highly dynamic, being substantially re‐modelled during growth and development. Cell walls determine size and shape of cells and contribute to the functional specialization of tissues and organs. Beyond the physiological dynamics, the wall structure undergoes changes upon biotic or abiotic stresses. In this review several cell wall traits, mainly related to pectin, one of the major matrix components, will be discussed in relation to plant development, immunity and industrial bioconversion of biomass, especially for energy production. Plant cell walls are a source of oligosaccharide fragments with a signalling function for both development and immunity. Sensing cell wall damage, sometimes through the perception of released damage associated molecular patterns (DAMPs), is crucial for some developmental and immunity responses. Methodological advances that are expected to deepen our knowledge of CW biology will also be presented.
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We have developed a non-invasive rapid and real-time red blood cell (RBC) hemolysis detection method which is a more accurate for point of care testing of hemolysis in various medical settings. An eight-parameter equivalent circuit is employed to quantify the release of hemoglobin (Hb) and the cytoplasm from RBC into the blood plasma. RBC hemolysis is induced by adding different volume fractions of distilled water into the blood. The cytoplasm released following RBC hemolysis is estimated from the experimental values. A strong relationship between RBC hemolysis and change in the electrical characteristics of blood has been demonstrated. The cytoplasm resistance (Rc) shows a linear relationship with the Hb. This relationship between Rc and Hb is described by the equation Rc = 0.2203Hb + 2.4775, with a correlation coefficient of 0.9905.
In order to understand the architecture of the primary plant cell wall, knowledge on its polysaccharides and their interactions is of importance. In this study, further architectural insight was obtained by sequential LiCl-DMSO and buffer extractions after planetary ball milling. After milling, up to 50% of all polysaccharides in the Chelating agent Unextractable Solids (ChUS) from carrot, tomato and strawberry solubilised in LiCl-DMSO without loss of structural information. Approximately 30% of all pectin was LiCl-DMSO insoluble but solubilised in the subsequent buffer extraction, and these populations had higher HG:RG-I ratios than LiCl-DMSO soluble populations. The degree of methyl-esterification (DM) of pure pectins highly determined its solubility in LiCl-DMSO. However, solubility of cell wall pectin was governed by more factors since both soluble and insoluble pectin were substantially methyl-esterified and acetylated. Digestion of LiCl-DMSO soluble and insoluble fractions by pectinases confirmed the presence of acetylated HG-regions for carrot and strawberry pectin.
In this study, distinguishing skin cancer cells (A431) and normal cells (HaCaT) was achieved using electrical impedance spectroscopy (EIS) with a novel developed device. The proliferation behaviors of the two cell types during a culture period of 5 days were characterized by the normalized impedance measured at 1465 Hz with simultaneous microscopic imaging for assistance. By fitting to the established equivalent circuits, A431 cells generated smaller resistance (Rc) values with smaller increasing variation, and comparable capacitance (Cc) values with similar decreasing variation compared with HaCaT cells. Moreover, Cc values were linearly correlated to the cell number. The results indicate that these two cell types can be distinguished with EIS based on the differences in the values and variation trends of Rc and Cc during the proliferation process in a real-time and label-free manner. Our work supplies a useful analytical approach for skin cancer cell research and may facilitate the early diagnosis of skin cancer.
Our understanding of plant biology is increasingly being built upon studies using 'omics and system biology approaches performed at the level of the entire plant, organ, or tissue. Although these approaches open new avenues to better understand plant biology, they suffer from the cellular complexity of the analyzed sample. Recent methodological advances now allow plant scientists to overcome this limitation and enable biological analyses of single-cells or single-cell-types. Coupled with the development of bioinformatics and functional genomics resources, these studies provide opportunities for high-resolution systems analyses of plant phenomena. In this review, we describe the recent advances, current challenges, and future directions in exploring the biology of single-cells and single-cell-types to enhance our understanding of plant biology as a system.
The detection of bacteria cells and their viability in food, water and clinical samples is critical to bioscience research and biomedical practice. In this work, we present a microfluidic device encapsulating a coplanar waveguide for differentiation of live and heat-killed Escherichia coli cells suspended in culture media using microwave signals over the frequency range of 0.5-20GHz. From small populations of ∼15 E. coli cells, both the transmitted (|S 21|) and reflected (|S 11|) microwave signals show a difference between live and dead populations, with the difference especially significant for |S 21| below 10GHz. Analysis based on an equivalent circuit suggests that the difference is due to a reduction of the cytoplasm conductance and permittivity upon cell death. The electrical measurement is confirmed by off-chip biochemical analysis: the conductivity of cell lysate from heat-killed E. coli is 8.22% lower than that from viable cells. Furthermore, protein diffusivity increases in the cytoplasm of dead cells, suggesting the loss of cytoplasmic compactness. These changes are results of intact cell membrane of live cells acting as a semipermeable barrier, within which ion concentration and macromolecule species are tightly regulated. On the other hand, the cell membrane of dead cells is compromised, allowing ions and molecules to leak out of the cytoplasm. The loss of cytoplasmic content as well as membrane integrity is measurable by microwave impedance sensors. Since our approach allows detection of bacterial viability in the native growth environment, it is a promising strategy for rapid point-of-care diagnostics of microorganisms as well as sensing biological agents in bioterrorism and food safety threats.
Comparing to conventional examinations, a continuous impedance analysis of single cells provide more detailed electrical characterization information about their pathological condition in a period. In this work, we present the 24-h observations of the electrical characteristics of single HeLa (human cervix adenocarcinoma) cells using impedance measurement and modeling method. A microfluidic device includes three-micropillars structure and measurement electrodes are used to single HeLa cell capture and impedance measurement, respectively. An electrical circuit comprising cytoplasm resistance, cell membrane capacitance, medium resistance, medium capacitance and electrodes resistance is used to obtain the variation on the location, shape and configuration of single HeLa cell. According to experiment and modeling results, cell approached the bottom of the microchannel at 3–5 h. At 5–15 h, the impedance characteristics of the single HeLa cell changed with the cell shape. After 15 h, cell membrane capacitance and cytoplasm resistance decreased due to cell membrane electroporation.
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Regulation of the mechanical properties of the cell wall is a key parameter used by plants to control the growth behavior of individual cells and tissues. Modulation of the mechanical properties occurs through the control of the biochemical composition and the degree and nature of interlinking between cell wall polysaccharides. Preferentially oriented cellulose microfibrils restrict cellular expansive growth, but recent evidence suggests that this may not be the trigger for anisotropic growth. Instead, non-uniform softening through the modulation of pectin chemistry may be an initial step that precedes stress-induced stiffening of the wall through cellulose. Here we briefly review the major cell wall polysaccharides and their implication for plant cell wall mechanics that need to be considered in order to study the growth behavior of the primary plant cell wall.
Secondary cell walls (SCWs) are produced by specialized plant cell types, and are particularly important in those cells providing
mechanical support or involved in water transport. As the main constituent of plant biomass, secondary cell walls are central
to attempts to generate second-generation biofuels. Partly as a consequence of this renewed economic importance, excellent
progress has been made in understanding how cell wall components are synthesized. SCWs are largely composed of three main
polymers: cellulose, hemicellulose, and lignin. In this review, we will attempt to highlight the most recent progress in understanding
the biosynthetic pathways for secondary cell wall components, how these pathways are regulated, and how this knowledge may
be exploited to improve cell wall properties that facilitate breakdown without compromising plant growth and productivity.
While knowledge of individual components in the pathway has improved dramatically, how they function together to make the
final polymers and how these individual polymers are incorporated into the wall remain less well understood.
Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.
Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.
The physical properties of biological materials impact their functions. This is most evident in plants where the cell wall contains each cell's contents and connects each cell to its neighbors irreversibly. Examining the physical properties of the plant cell wall is key to understanding how plant cells, tissues, and organs grow and gain the shapes important for their respective functions. Here, we present an atomic force microscopy-based nanoindentation method for examining the elasticity of plant cells at the subcellular, cellular, and tissue level. We describe the important areas of experimental design to be considered when planning and executing these types of experiments and provide example data as illustration.
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Conventional methods of plant cell analysis rely on growing plant cells in soil pots or agarose plates, followed by screening the plant phenotypes in traditional greenhouses and growth chambers. These methods are usually costly, need a large number of experiments, suffer from low spatial resolution and disorderly growth behavior of plant cells, with lack of ability to locally and accurately manipulate the plant cells. Microfluidic platforms take advantage of miniaturization for handling small volume of liquids and providing a closed environment, with the purpose of in vitro single cell analysis and characterizing cell response to external cues. These platforms have shown their ability for high-throughput cellular analysis with increased accuracy of experiments, reduced cost and experimental times, versatility in design, ability for large-scale and combinatorial screening, and integration with other miniaturized sensors. Despite extensive research on animal cells within microfluidic environments for high-throughput sorting, manipulation and phenotyping studies, the application of microfluidics for plant cells studies has not been accomplished yet. Novel devices such as RootChip, RootArray, TipChip, and PlantChip developed for plant cells analysis, with high spatial resolution on a micrometer scale mimicking the internal microenvironment of plant cells, offering preliminary results on the capability of microfluidics to conquer the constraints of conventional methods. These devices have been used to study different aspects of plant cell biology such as gene expression, cell biomechanics, cellular mechanism of growth, cell division, and cells fusion. This review emphasizes the advantages of current microfluidic systems for plant science studies, and discusses future prospects of microfluidic platforms for characterizing plant cells response to diverse external cues.
Plant stature and development are governed by cell proliferation and directed cell growth. These parameters are determined largely by cell wall characteristics. Cellulose microfibrils, composed of hydrogen-bonded β-1,4 glucans, are key components for anisotropic growth in plants. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes. In higher plants, these complexes are assembled into hexagonal rosettes in intracellular compartments and secreted to the plasma membrane. Here, the complexes typically track along cortical microtubules, which may guide cellulose synthesis, until the complexes are inactivated or internalized. Determining the regulatory aspects that control the behavior of cellulose synthase complexes is vital to understanding directed cell and plant growth and to tailoring cell wall content for industrial products, including paper, textiles, and fuel. In this review, we summarize and discuss cellulose synthesis and regulatory aspects of the cellulose synthase complex, focusing on Arabidopsis thaliana. Expected final online publication date for the Annual Review of Plant Biology Volume 65 is April 29, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
A method is described for purifying plant protoplasts from cellular and subcellular debris. The procedure utilizes a density buffer containing 9.6% sodium metrizoate and 5.6% Ficoll. The use of fluorescein diacetate for assessing the viability of plant protoplasts is also reported.
The process of cell wall regeneration around two species of higher plant protoplasts has been studied using reflection scanning electron microscopy. The first stage in the process is the formation of short fibres from randomly spaced centres. With protoplasts of tobacco leaf (Nicotiana tabacum L., cv White Burley) these fibres then elongate and interlace apparently at random to give rise to a matted continuous layer of wall. Protoplasts of a suspension culture of grapevine cells (Vitis vinifera L. cv Müller Thurgau) produce short fibres but these fail to elongate. Budding is observed during wall regeneration around vine protoplasts. The results are discussed in terms of the mechanical properties of the wall and its relationship to changes in plasmalemma morphology which are observed during wall formation.
This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells.
Dielectric spectroscopy or Electrochemical impedance spectroscopy (EIS) is traditionally used in corrosion monitoring, coatings evaluation, batteries, and electrodeposition and semiconductor characterization. However, in recent years, it is gaining widespread application in biotechnology, tissue engineering, and characterization of biological cells, disease diagnosis and cell culture monitoring. This article discusses the principles and implementation of dielectric spectroscopy in these bioanalytical applications. It provides examples of EIS as label-free, mediator-free strategies for rapid screening of biocompatible surfaces, monitoring pathogenic bacteria, as well as the analysis of heterogeneous systems, especially biological cells and tissues. Descriptions are given of the application of nanoparticles to improve the analytical sensitivities in EIS. Specific examples are given of the detection of base pair mismatches in the DNA sequence of Hepatitis B disease, TaySach's disease and Microcystis spp. Others include the EIS detection of viable pathogenic bacteria and the influence of nanomaterials in enhancing biosensor performance. Expanding applications in tissue engineering such as adsorption of proteins onto thiolated hexa(ethylene glycol)-terminated (EG6) self-assembled monolayer (SAM) are discussed.
Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.
Over the last century a number of techniques have been developed which allow the measurement of the dielectric properties of biological particles in fluid suspension. The majority of these techniques are limited by the fact that they only provide an average value for the dielectric properties of a collection of particles. More recently, with the advent of microfabrication techniques and the Lab-on-a-chip, it has been possible to perform dielectric spectroscopic experiments on single biological particles suspended in physiological media. In this paper we review current methods for single cell dielectric spectroscopy. We also discuss alternative single cell dielectric measurement techniques, specifically the ac electrokinetic methods of dielectrophoresis and electrorotation. Single cell electrical impedance spectroscopy is also discussed with relevance to a microfabricated flow cytometer. We compare impedance spectroscopy data obtained from measurements made using a microfabricated flow cytometer with simulation data obtained using an equivalent circuit model for the device.
This Account summarizes techniques for fabrication and applications in biomedicine of microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The methods and applications described focus on the exploitation of the physical and chemical properties of PDMS in the fabrication or actuation of the devices. Fabrication of channels in PDMS is simple, and it can be used to incorporate other materials and structures through encapsulation or sealing (both reversible and irreversible).
Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up approximately 90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of alpha-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.