Dead Yeast Cells Research Articles

Facile synthesis of carbon dots derived from ampicillin sodium for live/dead microbe differentiation, bioimaging and high selectivity detection of 2,4-dinitrophenol and Hg(II)

A highly photoluminescent small-size carbon dots (CDs) deriving ampicillin sodium were successfully obtained via one-step hydrothermal method. The prepared CDs showed uniform particle size and possessed good excitation-dependent characteristic. The dead bacteria and yeast cells could be selectively stained by the CDs, but the live ones not. It indicated that the CDs possessed a good potential for the live/dead bacteria and yeast differentiation. Cell and plant leaf imaging showed that the prepared CDs possessed low biotoxicity, excellent biocompatibility and multicolor fluorescence emission properties making it a good candidate for bioimaging application. The probe exhibited high selectivity and sensitivity to detect Hg(II) and 2,4-dinitrophenol (2,4-DNP), and excellent linear range of 0–50 μM for Hg(II) (R2 = 0.9920) and 0–25 μM for 2,4-DNP (R2 = 0.9919), with low detection limits of 33.40 nM and 13.44 nM for Hg(II) and 2,4-DNP, respectively. It revealed that dynamic quenching mechanism dominated in Hg(II) quenching the CDs fluorescent, while the quenching mechanism of 2,4-DNP detection belonged to the inner filter effect. The sensor was successfully applied to determine the Hg(II) and 2,4-DNP in real water samples. It shows that the fluorescent nanoprobe shows great promising potential for the detection of environmental pollutants.

Continuous Cell Characterization and Separation by Microfluidic Alternating Current Dielectrophoresis.

A novel alternating current (ac)-dielectrophoretic (DEP) microfluidic chip for continuous cell characterization and separation is presented in this paper. To generate DEP forces, two electrode-pads are embedded in a set of asymmetric orifices on the opposite sidewalls to produce the nonuniform electric fields. In the vicinity of a small orifice, the cells experience the strongest nonuniform gradient and are drawn toward it by the positive DEP forces, while the cells experiencing a negative DEP force are repelled away and move toward the large orifice. The DEP behaviors of yeast cells in suspending media with different ionic concentrations, i.e., different electrical conductivities, and over a large range of the ac electric field frequency were investigated. Furthermore, the lateral migrations of yeast cells as a function of the ac frequency were measured. The trends of measured lateral migrations of yeast cells are similar to the corresponding Clausius-Mossotti (CM) factors. In addition, by adjusting the frequency and strength of the ac electric field, the continuous separation of live and dead yeast cells as well as the yeast cells with targeted diameter and dielectric property can be easily achieved. This is the first time that the measurement of ac-DEP lateral migration of yeast cells in solutions with different electrical conductivities as a function of the applied frequency in a microfluidic chip was reported. This ac-DEP system provides a method to characterize the crossover frequency of the specific cells and manipulate the targeted cells.

Microencapsulation of beetroot pomace extractin cells of Saccharomyces cerevisiae

Beetroot pomace is an industrial by-product and ecological problem, as well as a potential rich source of natural antioxidants and pigments. In the present study, beetroot pomace extract (BPE) was encapsulated in non-plasmolysed, plasmolysed, and living Saccharomyces cerevisiae yeast cells by a freezedrying method. Living and dead yeast cells were microscopically assessed by using Neubauer chamber in order todetermine how the freeze-drying procedure affects changes in the yeast cell number. Total and surface phenolic contents were determined using the Folin-Ciocalteu method and encapsulation efficiency (EE) was calculated. The best EE was achieved in the case when BPE was encapsulated in living yeast cells. The successful encapsulation of BPE in yeast cells was confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). These results report the encapsulation of sensitive compounds in yeast cells by freeze-drying, showing that it is a good method for valuable compound preservation and has a potential use in food and pharmaceutical industries.

Microfluidic platform for dielectrophoretic separation of bio-particles using serpentine microelectrodes

Recently, there is a great concern in developing integrated microfluidic platforms for biological analysis systems that can perform general biochemistry and biological analysis. Dielectrophoresis, the movement of particles in a non-uniform electric field is widely used to separate viable and non viable cells with good efficiency. The present study aims to develop a universal bio-particle separator in a C-serpentine geometry which could separate particles by varying the operating conditions. We present the design, fabrication and testing for continuous separation structure based on dielectrophoretic forces. The C-Serpentine geometry is composed of consecutive C shaped curves to generate a gradient required for the dielectrophoretic effect. Known mixture of viable and non viable cells of Saccharomyces Cerevisiae was selectively trapped using negative dielectrophoretic force generated by microelectrodes. Through measurement of cell count, a trapping efficiency of 86% was obtained for mixed cell suspension and 93–98% trapping efficiency was obtained in case of trapping live and dead yeast cells individually. Also the results show that cell viability was not affected by the separation procedure as percentage of viable cells was calculated to be 96%.

Role of Debaryomyces hansenii yeast in improving the microbial and sensory properties of Monterey cheese

Debaryomyces hansenii yeast was grown in Malt Extract Broth medium at 30 °C for 5 days until the total count reached 5.6 ×108 colony forming units/ milliliter, Monterey cheese was made from cow's milk and adding of starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp.cremoris with D.hansenii yeast as adjunct starter in 2% for each treatment included M1 (100% starter bacteria), M2 (50% starter bacteria + 50% live D.hansenii) and M3 (50% starter bacteria + 50% dead D.hansenii),the ripening of Monterey cheese was done at 15°C and (85% relative humidity) for 56 days. The highest count of starter bacteria was 9.7 ×108 cfu/g in M1 treatment after 3 days of ripening, the lowest number of starter bacteria was 6.9 ×108 cfu/g in M2 treatment. The highest count of D.hansenii yeast was 4.3 x108 cfu/g after 3 days of ripening in M2 treatment, while the lowest count was 2.6 ×108 cfu/g in the same treatment after 56 days of ripening, no yeast was found in the treatments that had dead yeast cells, all treatments under study were decreased in the total count of coliform bacteria, Staphylococcus aureus and psychrotrophic bacteria after 14 days as they were within the standard specifications of these groups of microorganisms, Then reached to zero in the late time of ripening. M2 showed the best results of sensory evaluation in taste, flavor, textures, color and bitterness after 56 days of ripening.

Enhanced cell trapping throughput using DC-biased AC electric field in a dielectrophoresis-based fluidic device with densely packed silica beads.

This paper presents the use of DC-biased AC electric field for enhancing cell trapping throughput in an insulator-based dielectrophoretic (iDEP) fluidic device with densely packed silica beads. Cell suspension is carried through the iDEP device by a pressure-driven flow. Under an applied DC-biased AC electric field, DEP trapping force is produced as a result of non-uniform electric field induced by the gap of electrically insulating silica beads packed between two mesh electrodes that allow both fluid and cells to pass through. While the AC component is mainly to control the magnitude of DEP trapping force, the DC component generates local electroosmotic (EO) flow in the cavity between the beads and the EO flow can be set to move along or against the main pressure-driven flow. Our experimental and simulation results show that desirable trapping is achieved when the EO flow direction is along (not against) the main flow direction. Using our proposed DC-biased AC field, the device can enhance the trapping throughput (in terms of the flowrate of cell suspension) up to five times while yielding almost the same cell capture rates as compared to the pure AC field case. Additionally, the device was demonstrated to selectively trap dead yeast cells from a mixture of flowing live and dead yeast cells.

Dark-field microscope stool analysis - its role in diagnosis of yeast overgrowth in gut

Not long after birth, yeast, predominantly Candida albicans, colonizes the epithelium of oral cavity and the whole gastrointestinal tract. C. albicans lives in yeast, a non-harming form, as a commensal member of the microbial flora, but may turn into patho?gen infective form under certain conditions that encourage its overgrowth. In this phase, it may damage the intestinal wall and enter the bloodstream, causing invasive candidiasis with high mortality rate. It is essential to recognize candidaemia and start the lifesaving therapy on time. Recognizing the risk factors which allow candida to overgrow is the most important step in preventing candida?s overgrowth and chronic candidiasis, the previous status of invasive candidiasis. If this recognition is missed, and the overgrowth advances, a question remains how to discover and treat it and in which phase it should be done. A stool culture requires time and proves the presence of live yeast cells only. If the live yeast cells are not present in the stool, the result of the culture will be negative. In this paper, the author presents her experience of stool analysis under dark-field microscope, as a rapid, easy to carry out method for detecting the presence of live or dead yeast cells and yeast overgrowth.

Heat-Killed Yeast as a Pan-Fungal Vaccine.

Fungal infections continue to rise worldwide. Antifungal therapy has long been a mainstay for the treatment of these infections, but often can fail for a number of reasons. These include acquired or innate drug resistance of the causative agent, poor drug penetration into the affected tissues, lack of cidal activity of the drug and drug toxicities that limit therapy. In some instances, such as coccidioidal meningitis, therapy is life-long. In addition, few new antifungal drugs are under development. In light of this information a preventative vaccine is highly desirable. Although numerous investigators have worked toward the development of fungal vaccines, none have become commercially available for use in humans. In the course of our studies, we have discovered that heat-killed yeast (HKY) of Saccharomyces cerevisiae can be used as a vaccine and have shown that it has efficacy in the prevention and reduction of five different fungal infections when used experimentally in mice, which raises the possibility of a pan-fungal vaccine preparation. In our studies we grow S. cerevisiae in broth and heat-kill the organism at 70 ° C for 3 h. The number of dead yeast cells is adjusted and mice are vaccinated subcutaneously beginning 3-7 weeks prior to infection. After infection, efficacy is assessed on the basis of survival and residual burden of the fungus in the target organs. Alternatively, efficacy can be assessed solely on fungal burden at a predetermined time postinfection. Although itself it is unlikely to be moved toward commercialization, HKY can be used a positive control vaccine for studies on specific molecular entities as vaccines, and as a guidepost for the key elements of potential, more purified, pan-fungal vaccine preparations.

(Invited) Where Is Dielectrophoresis (DEP) Going?

Fifty years have passed since the first demonstration that DEP can be applied to the biomedical sciences. Pohl and Hawk [1] gave the first report of a purely physical technique being used to simultaneously distinguish and separate live and dead yeast cells. Over the past 50 years the theoretical aspects of DEP have attained a high level of refinement, whilst applications that include the manipulation of DNA, proteins, viruses, bacteria, blood cells, cancer cells and stem cells have been demonstrated. Stainless steel electrodes [1] used to generate the required high field gradients have been replaced by devices taking advantage of microfabrication and microfluidics, ranging from: microelectrode arrays [2]; insulator-based DEP [3, 4]; streaming DEP [5]; liquid electrodes [6]; carbon electrodes [7]. The key advances and relative advantages or otherwise of these various DEP techniques will be reviewed, with the aim to identify niche biomedical applications. 1. Pohl, H. A., Hawk, I. (1966) Separation of living and dead cells by dielectrophoresis, Science 152, 647-649. 2. Price, J. A. R., Burt, J. P. H., Pethig, R. (1988) Applications of a new optical technique for measuring the dielectrophoretic behaviour of micro-organisms, Biochim. Biophys. Acta 964, 221-230. 3. Masuda, S., Washizu, M., Nanba, T. (1989) Novel method of cell-fusion in field constriction area in fluid integrated circuit, IEEE Trans. Ind. Appl., 25, 732-737. 4. Chou, C-F., Tegenfeldt, J. O., Bakajin, O., Chan, S. S., Cox, E. C., Darnton, N., Duke, T., Austin, R. H. (2002) Electrodeless dielectrophoresis of single- and double-stranded DNA, Biophys. J., 83, 2170-2179. 5. Cummings, E. B., Singh, A. K. (2003) Dielectro-phoresis in microchips containing arrays of insulating posts: theoretical and experimental results, Anal. Chem., 75, 4724-4731. 6. Demierre, N., Braschler, T., Linderholm, P., Seger, U., van Lintel, H., Renaud, P. (2007) Characterization and optimization of liquid electrodes for lateral dielectro-phoresis, Lab Chip 7, 355-365. 7. Martinez-Duarte, R., Camacho-Alanis, F., Renaud, P., Ros, A. (2013) Dielectrophoresis of lambda-DNA using 3D carbon electrodes, Electrophoresis 34, 1113-1122.

The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 106 to 107CFU/mL and bacterial densities were approximately 108CFU/mL.

Using mixed inocula of Saccharomyces cerevisiae killer strains to improve the quality of traditional sparkling-wine

The quality of traditional sparkling-wine depends on the aging process in the presence of dead yeast cells. These cells undergo a slow autolysis process thereby releasing some compounds, mostly colloidal polymers such as polysaccharides and mannoproteins, which influence the wine’s foam properties and mouthfeel. Saccharomyces cerevisiae killer yeasts were tested to increase cell death and autolysis during mixed-yeast-inoculated second fermentation and aging. These yeasts killed sensitive strains in killer plate assays done under conditions of low pH and temperature similar to those used in sparkling-wine making, although some strains showed a different killer behaviour during the second fermentation. The fast killer effect improved the foam quality and mouthfeel of the mixed-inoculated wines, while the slow killer effect gave small improvements over single-inoculated wines. The effect was faster under high-pressure than under low-pressure conditions. Wine quality improvement did not correlate with the polysaccharide, protein, mannan, or aromatic compound concentrations, suggesting that the mouthfeel and foaming quality of sparkling wine are very complex properties influenced by other wine compounds and their interactions, as well as probably by the specific chemical composition of a given wine.

Enhancement of continuous-flow separation of viable/nonviable yeast cells using a nonuniform alternating current electric field with complex spatial distribution.

The variability in cell response to AC electric fields is selective enough to separate not only the cell types but also the activation states of similar cells. In this work, we use dielectrophoresis (DEP), which exploits the differences in the dielectric properties of cells, to separate nonviable and viable cells. A parallel-plate DEP device consisting of a bottom face with an array of micro-fabricated interdigitated electrodes and a top face with a plane electrode was proposed to facilitate the separation of cells by creating a nonuniform electric field throughout the flow channel. The operation and performance of the device were evaluated using live and dead yeast cells as model biological particles. Further, numerical simulations were conducted for the cell suspensions flowing in a channel with a nonuniform AC electric field, modeled on the basis of the equation of motion of particles, to characterize the separation efficiency by changing the frequency of applied AC voltage. Results demonstrated that dead cells traveling through the channel were focused onto a site around the minimum electric field gradient in the middle of the flow stream, while live cells were trapped on the bottom face. Cells were thus successfully separated under the appropriately tuned frequency of 1 MHz. Predictions showed good agreement with the observation. The proposed DEP device provides a new approach to, for instance, hematological analysis or the separation of different cancer cells for application in circulating tumor cell identification.

Fifty years of dielectrophoretic cell separation technology.

In 1966, Pohl and Hawk [Science 152, 647-649 (1966)] published the first demonstration of dielectrophoresis of living and dead yeast cells; their paper described how the different ways in which the cells responded to an applied nonuniform electric field could form the basis of a cell separation method. Fifty years later, the field of dielectrophoretic (DEP) cell separation has expanded, with myriad demonstrations of its ability to sort cells on the basis of differences in electrical properties without the need for chemical labelling. As DEP separation enters its second half-century, new approaches are being found to move the technique from laboratory prototypes to functional commercial devices; to gain widespread acceptance beyond the DEP community, it will be necessary to develop ways of separating cells with throughputs, purities, and cell recovery comparable to gold-standard techniques in life sciences, such as fluorescence- and magnetically activated cell sorting. In this paper, the history of DEP separation is charted, from a description of the work leading up to the first paper, to the current dual approaches of electrode-based and electrodeless DEP separation, and the path to future acceptance outside the DEP mainstream is considered.

(Invited) More Than Moore Applications with Thin-Film-Transistors Array Substrates from Liquid Cristal Display: New Devices for Biological Cells Analyses

Thin-Film-Transistors technology has been a famous technology since 20 years for the development of Liquid-Cristal-Displays, for televisions, smart-phones or video games screens applications. Thanks to that technology, a dense array of pixels, individually controllable by an array of independent transistors, can be achieved, allowing screens with very high resolution (pixels of some ten micrometers square). The key point is the fabrication of the array of transistors by deposition technique on a substrate which are usually glass or transparent plastic. The cross-section of a TFT-LCD screen shows that it consists into three different layers attached together. The upper layer (or upper glass) is a glass covered on one side with a polarizer and on the other side, with a color filter. This one is in contact with the second layer which is the liquid crystal layer. The third layer (or lower glass) is a glass covered with a large and dense array of independent and transparent micro-electrodes (made in Indium Tin Oxide, ITO), individually controllable by the array of independant transistors. That lower glass is called here “TFT-substrate” and is the key device in the research described hereafter.In parallel to the development of TFT technology, since about 30 years, micro-technology has seen tremendous headways thanks to the development of micro-fluidics and bio-microsystems. Bio-microsystems require transparent substrates, for the ease of optical observation through the device, and micro-fluidics to confine the liquid environment usually necessary. More recently, the integration of some electronic components, from simple micro-electrodes to more complex ISFET (Ion-Sensitive Field Effect Transistors) have widened the frame of applications in that area. However, in such devices, alignment between the electronic component and the biological entity remains a key issue and is tricky. A large and dense array of independent electronic components would avoid to care about alignment, but standard microtechnology hardly allow that solution. The solution can be brought by the TFT technology, as it provides such an array of independent electronic components. Such substrate becomes a very interesting device for biological applications, as it will be demonstrated in this talk.The TFT technology, at first mainly applied to LCDs, diversifies its applications in a “More than Moore” objective, proposing new and unique solutions for biological purposes: an electrically controllable transparent substrate. In addition, the transistor array, initially used only as simple switches to control the electrodes ON and OFF state, becomes an active part in the device, allowing not only DC or AC signal application, but also sensing, either through the micro-electrodes, or directly being themselves a possible array of ISFETs.Our group aims at demonstrating the possible applications, as well as the limitations, of TFT-substrates in the field of biological cell research. The structure of the TFT-substrate is reported in Figure, as well as some experimental results. The substrate has been obtained by detaching by hand the upper glass from the lower glass of a smart phone screen, and then by cleaning it with organic cleaning, to remove the liquid crystal. Each micro-electrode is connected to the drain of each transistor. The sources of the TFTs are connected line by line to the pad of the corresponding line. The gates of the TFTs are connected column by column to the pad of the corresponding column. When one source line and one gate line are ON, the TFT at its intersection, as well as the attached electrode becomes ON. Usual TFT devices are used under DC potential. However, for biological purposes, AC potential is needed. Characterization of the output signal at the drain (the micro-electrode), when a DC voltage is applied at the gate and an AC voltage at the source, has been performed. It showed an attenuation of the output signal with the decreasing gate and an attenuation of -20dB for each decade of the frequency above 1 kHz. Despite these attenuations, several results have already been successfully obtained. Dielectrophoresis on micro-beads and on yeast cells have demonstrated a negative dielectrophoretic force on micro-beads while a positive dielectrophoretic force was obtained on yeast cells at 100 kHz. Sensing was also performed using the same substrate. Impedance measurements on yeast cells was done, showing that the impedance between two adjacent micro-electrodes was increasing, as expected, with the number of cells, at a frequency of 10 kHz. Impedance measurements were also performed on live and dead yeast cells, showing a clear decreasing of impedance, as expected, in the case of dead cells. pH sensing was also performed. Other experiments are under investigation for more dedicated purposes. Figure 1

The application of flow cytometry in microbiological monitoring during winemaking: two case studies

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometry.

10114 マイクロチャンネルによるイースト菌の電気特性計測

The demand of high-speed and efficient cell separation techniques has increased with the increased interest in regenerative medicine. Conventional methods of cells separation are not so efficient due to the ID operation and high load toward cells. In this paper, the possibility of peculiar cell separation with dielectrophoretic (DEP) force is studied and discussed. The opposite DEP forces for living and dead yeast cells are found at the applied frequency near 4MHz. Moreover, the DEP force simulations is performed based on the complex dielectric constant obtained from experiments. From the simulation result, separation rate of living cells and dead cells are predicted to be 65% and 90%, respectively. Finally, we concluded that it is possible to separate peculiar cells with DEP force.

The contribution of glutathione to the destabilizing effect of yeast on wheat dough

Any factor which impairs the development of the gluten network affects the gas retention capacity and the overall baking performance. This study aimed to examine why rising yeast concentrations (Saccharomyces cerevisiae) decrease the dough elasticity in an asymptotic manner. Since in 27 commercial fresh and dry yeasts up to 81mg glutathione (GSH) per 1g dry sample were found. Through the addition of reduced GSH in dough without yeast, the extent of dough weakening was analysed. Indeed rheological measurements confirmed that yeast-equivalent levels of GSH had a softening effect and during 3h fermentation the weakening coefficient increased from 0.3% to 20.4% in a Rheofermentometer. The present results indicate that free –SH compounds, as represented by GSH, considerably contribute to the softening of dough through dead yeast cells.

Rapid Purification of Glycerol by-product from Biodiesel Production through Combined Process of Microwave Assisted Acidification and Adsorption via Chitosan Immobilized with Yeast

Biodiesel is a proven alternative to the petroleum diesel fuel. During biodiesel production, glycerol is produced as a by-product. This by-product consist of impureties such as soap, salts, sodium catalyst and so on. Traditionally, two of the most conventional techniques that is applied to glycerol purification are distillation and ion-exchange. These techniques are, however, still expensive to generate pure glycerol. Recently, several alternative “combination” treatment procedures have been used. These treatment has several advantages over others methods such as producing large amounts of glycerol-rich layer that requires simple treatments and not causing any high operational cost. In this study, the combination treatment process have been used in order to reach high glycerol content. Basically, these stages starts with using microwave assisted acidification process and the next process utilizing a bioadsorbent synthesized from dead yeast cells immobilized on chitosan. The final yield of glycerol was about 93.1-94.2% (w/w).

Analysis of Normal and Infected Bio-Cell Using Dual Nanoprobe

The objective of this paper is to analyze the yeast cell, liver cell and blood cell under both infected and healthy conditions by applying electrical signal through dual nanoprobe from source to cell. Knowledge of nanoprobe based bio-cell analysis can be used to differentiate infected cells from healthy ones, since their electrical behaviour is different. The voltage can be applied either inside the cytoplasm penetrating the cell wall, or at the outer surface of cell membrane. In this paper, the simulation has been carried out by applying voltage inside the cytoplasm using ABAQUS 6.10 CAE, powerful finite element software and the results obtained from simulation shows current flow healthy yeast and dead yeast cells are 1·9nA and 34pA respectively whereas the value of the current measured for the leukaemia affected blood cell is 21·2nA, being 2% less than the White Blood Cell (WBC). Since the conductivity of the cytoplasm of the healthy cell is theoretically higher than that of dead or infected cell, which is verified by simulation. The results from simulation show that the measured cell current from liver tumour cell is 2-7 times larger than healthy liver cell including membrane as it is supposed to be since the conductivity of cell including cell membrane is theoretically greater for dead or infected cell than healthy cell.

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip.

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.