Nonviable Yeast Cells Research Articles

Design and optimization of microchannel for enhancement of the intensity of induced signal in particle/cell impedance measurement.

The measured impedance signal of a single particle or cell in a microchannel is of the μA level, which is a challenge for measuring such weak signals. Therefore, it is necessary to improve the intensity for expanding the applications of impedance measurement. In this paper, we analyzed the impact of geometric parameters of microchannel on output signal intensity by using the three-dimensional finite element method. In comparison to conventional microchannels, which are distributed at a uniform height, the microchannels in this design use the height difference to enhance the signal intensity. By analyzing the effects of the geometric dimensions of the constriction channel, main channel height, radius of particles, types of cells, shapes of particles with different ellipticities, and particles spacing on the current signal, we concluded the optimal dimensions of these parameters to improve the intensity of the induced current signal. Through the fabrication of the optimized size of device and experimental demonstration, it is verified that the current signal intensity caused by the particle with a diameter of 10 µm is nearly twice that of the conventional structure with a height of 20 µm, which proves the correctness of the optimization results and the feasibility of this work. In addition, the performance of the device was verified by measuring the mixtures of different size particles as well as non-viable and viable yeast cells.

Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

Label-Free Multitarget Separation of Particles and Cells under Flow Using Acoustic, Electrophoretic, and Hydrodynamic Forces.

Multiplex separation of mixed biological samples is essential in a considerable portion of biomedical research and clinical applications. An automated and operator-independent process for the separation of samples is highly sought after. There is a significant unmet need for methods that can perform fractionation of small volumes of multicomponent mixtures. Herein, we design an integrated chip that combines acoustic and electric fields to enable efficient and label-free separation of multiple different cells and particles under flow. To facilitate the connection of multiple sorting mechanisms in tandem, we investigate the electroosmosis (EO)-induced deterministic lateral displacement (DLD) separation in a combined pressure- and DC field-driven flow and exploit the combination of the bipolar electrode (BPE) focusing and surface acoustic wave (SAW) sorting modules. We successfully integrate four sequential microfluidic modules for multitarget separation within a single platform: (i) sorting particles and cells relying on the size and surface charge by adjusting the flow rate and electric field using a DLD array; (ii) alignment of cells or particles within a microfluidic channel by a bipolar electrode; (iii) separation of particles based on compressibility and density by the acoustic force; and (iv) separation of viable and nonviable cells using dielectric properties via the dielectrophoresis (DEP) force. As a proof of principle, we demonstrate the sorting of multiple cell and particle types (polystyrene (PS) particles, oil droplets, and viable and nonviable yeast cells) with high efficiency. This integrated microfluidic platform combines multiple functional components and, with its ability to noninvasively sort multiple targeted cells in a label-free manner relying on different properties, is compatible with high-definition imaging, showing great potential in diverse diagnostic and analysis applications.

Вплив температури на зброджування високогустинного сусла

Значне збільшення вартості енергоресурсів сприяло розвитку сучасних енергоощадних технологій. Для виготовлення пива затрачається велика кількість енергії та теплоносіїв на стадії приготування сусла та холодоагентів на стадії бродіння. В умовах енергетичної кризи та великої конкуренції серед виробників виникає потреба запроваджувати нові, енергоощадні технології, зокрема високогустинне пивоваріння. Потенційні переваги технології високогустинного пивоваріння над звичайною технологією включають також значну економію води, більшу кількість спирту і зниження витрат енергії та праці, менші капітальні витрати. Проте, переваги високогустинного пивоваріння стають очевидними, коли тривалість бродіння залишається помірною, а життєздатність дріжджів та смак пива є адекватними. Ці показники в значній мірі залежать від параметрів процесу головного бродіння, зокрема від температури. В роботі досліджено вплив температури головного бродіння 9, 12, 15 і 18 °С на зброджування високогустинного сусла концентрацією 16% сухих речовин та фізіологічні властивості дріжджів. Для досліджень використовували дріжджі низового бродіння раси W-34/70. Тривалість бродіння становила 9 діб. Встановлено, що при традиційній температурі бродіння 9 °С процес відбувається повільно та припиняється передчасно. Проведення бродіння при 18 °С дозволяє досягти високого ступеня зброджування, проте погіршуються фізіологічні властивості дріжджів, зокрема знижується вміст клітин з глікогеном та погіршуються флокуляційні властивості дріжджів. Температура бродіння практично не впливала на вміст нежиттєздатних дріжджових клітин. Проте їх кількість була дещо вищою від значення, якого дотримуються на пивоварних підприємствах, що зумовлено впливом осмотичного та етанольного стресу. Оптимальна температура головного бродіння повинна знаходитись в межах 12–15 °С. За цих значень температур досягається висока бродильна активність дріжджів, ступінь зброджування пива та зберігаються хороші фізіологічні властивості дріжджів, а вміст віцинальних дикетонів не перевищує гранично-допустимої концентрації для молодого пива.

Survival of probiotic Lactobacillus acidophilus in acidic environment is enhanced in the presence of Saccharomyces cerevisiae

A number of health benefits have been proved for probiotic bacteria by many studies and probiotics are increasingly incorporated into foods. However, these market preparations have shown low viability of probiotics in human digestion. Therefore, providing viable probiotic cells to the colon and maintaining their metabolic activity against severe conditions of human digestion are increasingly interested by many recent scientific researches. In this trend, our research showed that by creating a physical barrier, the presence of Saccharomyces cerevisiae (SC) in Lactobacillus acidophilus (LA) suspension can effectively protect probiotic cells from stresses of digestion. After 150 minutes in simulated gastric juice, the survival of LA is significant improved (p<0.05) by forming cell-cell contact with SC cells. The LA-only cells show that most cells die with viability of 0% due to low pH medium, compared with 11.025 ± 1.127% of LA+SC mixture. Besides, we found that the cell concentration ratio at 1:10 between SC and LA cells performs highest protective effects on the probiotic in the acidic environment with 10.122 ± 1.348% LA viability. This concentration ratio is the critical value because when the SC concentration is increasingly higher (SC÷LA concentration ratios higher than 1÷10), LA viability shows no significantly different increase. We also found that yeast cells with oxidized carbohydrates on cell’s surface have many adverse impacts on co-aggregation (4.003 ± 0.115% after 240-minute treatment) while non-viable yeast cells with damaged and denatured protein on cell’s surface still maintains a high percentage of co-aggregation with LA (26.050 ± 0.259% after 240-minute treatment).

Design and simulation of a MEMS based cell separator utilizing 3D travelling-wave dielectrophoresis

In this work a novel electrode structure is proposed to separate viable and non-viable yeast cells by travelling-wave dielectrophoresis (twDEP). The electrodes are chevron-shaped which the sharp angle at the center of the electrodes is replaced by a curvature. We employed finite element method to simulate and observe the electric fields related to conventional dielectrophoresis (cDEP) and twDEP and correspondingly to estimate the cell trajectories under the applied electric field. Furthermore, we discuss different physical and geometrical parameters to design our twDEP microseparator to get the most accurate operation and higher separation efficiency. First, we study the adequate frequency and medium conductivity. Then, we explore the optimized electrode dimensions for target cells. From the simulations, we discovered that by the proposed electrode structure and applying phase shifted AC electric potential with amplitude of 2 V p-p and frequency of 70 kHz viable yeast cells discriminate from non-viable ones and get focused on a band at the center of the channel, simultaneously. The simulation results reveal that here the maximum twDEP force is applied to yeast cells when electrode width is about 12 μm. Finally, we propose possible different electrode forms that can be derived from our proposed electrode shape to manipulate different cells/particles.

Saccharomyces cerevisiae EC-1118 enhances the survivability of probiotic Lactobacillus rhamnosus HN001 in an acidic environment.

The present study attempted to partially characterize and elucidate the viability-enhancing effect of a yeast strain Saccharomyces cerevisiae EC-1118 on a probiotic strain Lactobacillus rhamnosus HN001 under acidic conditions using a model system (non-growing cells). The yeast was found to significantly enhance (P < 0.05) the viability of the probiotic strain under acidic conditions (pH 2.5 to 4.0) by 2 to 4 log cycles, and the viability-enhancing effects were observed to be influenced by pH, and probiotic and yeast concentrations. Microscopic observation and co-aggregation assay revealed that the viability-enhancing effect of the yeast could be attributed to direct cell-cell contact co-aggregation mediated by yeast cell surface and/or cell wall components or metabolites. Furthermore, non-viable yeast cells killed by thermal means were observed to enhance the viability of the probiotic strain as well, suggesting that the surface and/or cell wall component(s) of the yeast contributing to co-aggregation was heat-stable. Cell-free yeast supernatant was also found to enhance the viability of the probiotic strain, indicating the presence of protective yeast metabolite(s) in the supernatant. These findings laid the foundation for further understanding of the mechanism(s) involved and for developing novel microbial starter cultures possibly without the use of live yeast for ambient-stable high-moisture probiotic foods.

Quantitative characterization for dielectrophoretic behavior of biological cells using optical tweezers

We report a method to precisely quantify dielectrophoretic (DEP) forces and cutoff frequencies (fc) of viable and nonviable yeast cells. The method consists of a two-step process in which generated DEP forces act upon a cell through a micro-electrode device, followed by direct measurement of DEP forces using optical tweezers. DEP behaviors of viable and nonviable yeast cells are monitored as a function of AC frequency. We believe that the proposed method can be used as a powerful platform for cell-based assays to characterize the DEP behavior of various cell types including cancer and normal cells.

Modifying Dielectrophoretic Response of Nonviable Yeast Cells by Ionic Surfactant Treatment

Nonviable cells are essential biosystems, due to the functionalities they offer and their effects on viable cells. Therefore, the separation and immobilization of nonviable cells separately or in the vicinity of viable cells is of great importance for many fundamentals investigations in cell biology. However, most nonviable cells become less polarizable than the surrounding medium at conductivities above 0.01 S/m. This means that in such a medium, dielectrophoresis, despite its great versatilities for manipulation of cells, cannot be employed for immobilizing nonviable cells. Here, we present a novel approach to change the dielectrophoretic (DEP) response of nonviable yeast cells by treating them with low concentrations of ionic surfactants such as sodium dodecyl sulfate. After this treatment, they exhibit a strong positive DEP response, even at high medium conductivities. The capability of this treatment is demonstrated in two proof-of-concept experiments. First, we show the sorting and immobilization of viable and nonviable yeast cells, along consecutive microelectrode arrays. Second, we demonstrate the immobilization of viable and nonviable cells in the vicinity of each other along the same microelectrode array. The proposed technique allows DEP platforms to be utilized for the immobilization and subsequent postanalysis of both viable and nonviable cells with and without the presence of each other.

Electronic detection of dielectrophoretic forces exerted on particles flowing over interdigitated electrodes.

Dielectric particles flowing through a microfluidic channel over a set of coplanar electrodes can be simultaneously capacitively detected and dielectrophoretically (DEP) actuated when the high (1.45 GHz) and low (100 kHz-20 MHz) frequency electromagnetic fields are concurrently applied through the same set of electrodes. Assuming a simple model in which the only forces acting upon the particles are apparent gravity, hydrodynamic lift, DEP force, and fluid drag, actuated particle trajectories can be obtained as numerical solutions of the equations of motion. Numerically calculated changes of particle elevations resulting from the actuation simulated in this way agree with the corresponding elevation changes estimated from the electronic signatures generated by the experimentally actuated particles. This verifies the model and confirms the correlation between the DEP force and the electronic signature profile. It follows that the electronic signatures can be used to quantify the actuation that the dielectric particle experiences as it traverses the electrode region. Using this principle, particles with different dielectric properties can be effectively identified based exclusively on their signature profile. This approach was used to differentiate viable from non-viable yeast cells (Saccharomyces cerevisiae).

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

Particle Focusing and Separation through Lab-On-Chip Device by Dielectrophoresis

A Lab-On-Chip micro-device has been introduced for the focusing and separation of particles which exploits negative dielectrophoretic (nDEP) force. A 3D numerical computational model was presented to simulate the focusing and separation process. In particular, the model will solve for bioparticle movement and Joule heating. In the experimental study, the mixture of viable and nonviable yeast cells was used to demonstrate the focusing and separation effect of the designed microfluidic device. The fabricated chip performs a desired focusing result for the conditions with the electric fields frequency of 1MHz, the medium conductivity of 0.01S/m and the applied voltage V = 20V pp . The mixture of viable and nonviable yeast cells were focused to the central plane of the micro-channel and then separated by non-equilibrium nDEP induced by the single part of the last pair of electrodes with conductivity σ = 0.01S/m, applied voltage V = 20V pp and applied electric field frequency of 5Mhz, which validities the prediction of the proposed 3D model. This Lab-On-Chip device provides a simple and effective mean of particle focusing.

Particle Focusing through Lab-on-Chip Device by Negative Dielectrophoresis

In this paper a Lab-On-Chip device using negative dielectrophoresis has been proposed for the focusing of particles at the center of the micro-channel. A 3D computational model was presented to simulate the focusing process. In particular, the model will solve for bioparticle movement and Joule heating. In the experimental study, the mixture of viable and nonviable yeast cells was used to demonstrate the focusing effect of the designed microfluidic device. The fabricated chip performs a desired focusing result for the conditions with the electric fields frequency of 1MHz, the medium conductivity of and the applied voltage , which validities the proposed 3D computational model. This Lab-On-Chip device provides a simple and effective mean of particle focusing.

Distinguishing the viability of a single yeast cell with an ultra-sensitive radio frequency sensor

We propose and demonstrate a simple, ultra sensitive radio frequency (RF) sensor to detect a single yeast cell and distinguish its viability in a microfluidic channel. On-chip interference is used to cancel background probing signals to improve sensor sensitivity. Individual viable and nonviable yeast cells (approximately 5.83 +/- 0.85 microm in diameter) are measured with clear sensing and identification of these cells.

Glutaraldehyde enhanced dielectrophoretic yeast cell separation

We introduce a method for improved dielectrophoretic (DEP) discrimination and separation of viable and nonviable yeast cells. Due to the higher cell wall permeability of nonviable yeast cells compared with their viable counterpart, the cross-linking agent glutaraldehyde (GLT) is shown to selectively cross-link nonviable cells to a much greater extent than viable yeast. The DEP crossover frequency (cof) of both viable and nonviable yeast cells was measured over a large range of buffer conductivities (22 muScm-400 muScm) in order to study this effect. The results indicate that due to selective nonviable cell cross-linking, GLT modifies the DEP cof of nonviable cells, while viable cell cof remains relatively unaffected. To investigate this in more detail, a dual-shelled oblate spheroid model was evoked and fitted to the cof data to study cell electrical properties. GLT treatment is shown to minimize ion leakage out of the nonviable yeast cells by minimizing changes in cytoplasm conductivity over a large range of ionic concentrations. This effect is only observable in nonviable cells where GLT treatment serves to stabilize the cell cytoplasm conductivity over a large range of buffer conductivity and allow for much greater differences between viable and nonviable cell cofs. As such, by taking advantage of differences in cell wall permeability GLT magnifies the effect DEP has on the field induced separation of viable and nonviable yeasts.

Dielectrophoretic field-flow method for separating particle populations in a chip with asymmetric electrodes.

This paper presents a field-flow method for separating particle populations in a dielectrophoretic (DEP) chip with asymmetric electrodes under continuous flow. The structure of the DEP device (with one thick electrode that defines the walls of the microfluidic channel and one thin electrode), as well as the fabrication and characterization of the device, was previously described. A characteristic of this structure is that it generates an increased gradient of electric field in the vertical plane that can levitate the particles experiencing negative DEP. The separation method consists of trapping one population to the bottom of the microfluidic channel using positive DEP, while the other population that exhibits negative DEP is levitated and flowed out. Viable and nonviable yeast cells were used for testing of the separation method.

T Helper 1–Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1–stimulating adjuvant in P. brasiliensis–infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.

Sequential Field-Flow Cell Separation Method in a Dielectrophoretic Chip With 3-D Electrodes

This paper presents a sequential dielectrophoretic field-flow separation method for particle populations using a chip with a 3-D electrode structure. A unique characteristic of our chip is that the walls of the microfluidic channels also constitute the device's electrodes. This property confers the opportunity to use the electrodes' shape to generate not only the electric field gradient required for dielectrophoretic force but also a fluid velocity gradient. This interesting combination gives rise to a new solution for the dielectrophoretic separation of two particle populations. The proposed sequential field-flow separation method consists of four steps. First, the microchannel is filled with the mixture of the two populations of particle. Second, the particle populations are trapped in different locations of the microfluidic channels. The population, which exhibits positive dielectrophoresis (DEP), is trapped in the area where the distance between the electrodes is the minimum, while the other population that exhibits negative DEP is trapped in locations of maximum distance between electrodes. In the next step, increasing the flow in the microchannels will result in an increased hydrodynamic force that sweeps the cell population trapped by positive DEP out of the chip. In the last step, the electric field is removed, and the second population is swept out and collected at the outlet. For theoretical and experimental exemplification of the separation method, a population of viable and nonviable yeast cells was considered.

Dielectrophoresis microsystem with integrated flow cytometers for on‐line monitoring of sorting efficiency

Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DEP. The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements (waveguide, lens, and fiber coupler). By comparing the cell counting rates measured by the two FCs, the collection efficiency of the DEP filter can be determined. The chips were used for quantitative determination of the effect of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency. A theoretical model for the capture efficiency was developed and a reasonable agreement with the experimental results observed. Viable and non-viable yeast cells showed different frequency dependencies and were sorted with high efficiency. At 2 MHz, more than 90% of the viable and less than 10% of the non-viable cells were captured on the DEP filter. The presented approach provides quantitative real-time data for sorting a large number of cells and will allow optimization of the conditions for, e.g., collecting cancer cells on a DEP filter while normal cells pass through the system. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications.

A High-Throughput 3-D Composite Dielectrophoretic Separator

Dielectrophoresis has great potential to offer a range of diverse fields from bioprocessing to clinical medicine, but is hampered by low throughput rates to the micrometer scale of the electrodes required to generate highly nonuniform fields. Here we describe a novel approach to electrode construction, using a drilled laminated structure to form channels bearing electrodes 30 microm across and 150 microm apart. Since these electrodes appear along all sides of the drilled bore, the trapping efficiency is improved over conventional devices. We have developed and demonstrated a separator capable of sorting a 50:50 mixture of viable and nonviable yeast cells into an 86:14 mixture at 25 mLhr(-1).