基于微流体数字化技术的流式细胞术的设计

针对流式细胞术的核心之一——单细胞的液流驱动系统,提出了基于微流体数字化驱动-控制技术实现单细胞排列及输出的方法,分析了单细胞流的驱动机理,通过对流动室施加数字化可编码的高频驱动脉冲,细胞从连续的鞘液流中分割成单细胞流输出。设计了一种流式细胞仪核心部分石英微流动室,并采用石英玻璃精细加工而成。进行了人体外周血淋巴细胞的驱动实验研究,可通过荧光显微镜实时观测微通道内的单细胞流动情况,方法简单,操作方便。     

Fully automated on-chip imaging flow cytometry system with disposable contamination-free plastic re-cultivation chip

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 10(6) particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.     

Role of flow cytometry for the improvement of bioprocessing of oleaginous microorganisms

Oleaginous microorganisms, such as yeast, fungi, microalgae and bacteria, represent a key segment of second generation feed-stock materials and are considered to synthesize a wide range of industrially important chemical compounds. Oleaginous microorganisms possess a broad varieties of chemical compounds such as carotenoids, pigments, carbohydrates, chlorophyll, and storage-material lipids. Oleaginous microorganisms have been recognized as promising sources for the synthesis of unsaturated, especially polyunsaturated fatty acids (PUFA). So far, a variety of high-throughput screening methodologies (HTMs) have been employed for the development of bioprocessing of oleaginous microorganisms for sustainable production of industrially valuable compounds. Of HTMs, flow cytometry (FC) and sorters (FACS) have received substantial interest as better HTMs because of their ability to screen large numbers of cells within seconds, and interrogate and isolate living cells at single-cell level. Forward and side scattering signals of FC are used to determine the physiological state of the cell while different channels available in the FC facilitate the detection of signals produced from fluorophores. Simultaneous measurement of physiological characteristics along with specific compound accumulation at single-cell level enables the possibility of separating a particular phenotype with specific properties from a population. Different microbial strain development strategies in combination with FACS produced improved phenotypes with desired properties. This review first summarizes the FACS methodologies suitable for oleaginous microorganisms and the significant progress that has been achieved in oleaginous microorganisms using FACS, and highlights the important, advanced and future prospects of FACS methodologies that are suitable for the development of bioprocessing in oleaginous microorganisms. © 2018 Society of Chemical Industry     

Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.     

Hydrogenation reactions catalyzed by colloidal palladium nanoparticles under flow regime

A glycerol colloidal solution of palladium nanoparticles stabilized by PVP (PdPVP, mean diameter of 2.2 ± 0.8 × 10⁻⁹ m) was employed under flow conditions for hydrogenation reactions. After optimization, we selected the tube-in-tube flow reactor as the more suitable system for this catalytic process due to its ability to regulate the addition of hydrogen. PdPVP nanoparticles were synthesized and fully characterized ((HR)-TEM, PXRD, EA, and ICP) both in glycerol solution and at solid state. The reduction of different substrates, including nitrobenzene, 4-phenylbut-3-en-2-one, and isophorone, was achieved under relatively smooth conditions (5–7 × 10⁵ Pa of H₂) in short times (<600 s). Unfortunately, the reduction of 1-dodecene was not possible due to the faster isomerization reaction. The optimal effusion of hydrogen in the colloidal phase together with the catalytic efficiency of this system is noteworthy.     

Toxicity of Carbon Nanomaterials—Towards Reliable Viability Assessment via New Approach in Flow Cytometry

The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C₃N₄) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C₃N₄ and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.     

Effects of pluronic F68 micellization on the viability of neuronal cells in culture

Triblock copolymers with surface‐active properties, referred to as Pluronic, have shown potential medical applications such as drug delivery to selective targets in the human body. In particular, the transport of anti‐inflammatory substances to the brain is required for illness treatment, thus the study of delivery agents that cross the blood–brain barrier is relevant. In this article we study the effects of the micelle formation on the morphologic and cytotoxic properties of Pluronic F68. We determinate the critical micellar concentration (CMC) by standard tensiometric and absorbance measurements, and also we analyze the morphology of polymers by atomic force microscopy. Our observations indicate that the morphological properties of F68 are drastically modified in the CMC range, as well as the ability to increase the viability of neuroblastoma cells maintained under culture conditions, as compared with nontreated cells. Our conclusions highlight the close correlation between morphological and physiochemical properties of Pluronic, which must be further understood in order to achieve highly controlled pharmacological uses. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2159–2164, 2013     

Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.     

Influence of hydroxyvalerate composition of polyhydroxy butyrate valerate (PHBV) copolymer on bone cell viability and in vitro degradation

The objective of this study was to elucidate the role of hydroxyvalerate (HV) composition in polyhydroxy butyrate valerate (PHBV) copolymer film on the degradation of copolymer and osteoblastic cell activity. Degradation was studied by monitoring time‐dependent changes in mass and chemical composition of the macroporous films. The mass loss of PHBV film upon 19 weeks of exposure to pH 7.4 phosphate buffer medium was found to range from 2.8% to 9.2% with a strong dependence on the original composition of the copolyester film and morphology. Tapping mode atomic force microscopy (TMAFM) was used to examine the roughness change of polyester films due to exposure to buffer medium. Chemical analysis of the degraded film was carried out using NMR to aid in the interpretation of the mass loss and TMAFM data. The NMR results showed a significant decrease in the mol % of HV content in the degraded PHBV film. Additionally, we established that UMR‐106 cell proliferation on macroporous PHBV matrix is minimally enhanced by the HV content of PHBV copolymer. Information provided by this study can be used in the selection of appropriate PHBV copolymer for clinical use where the biopolymer needs to remain physically intact and chemically unchanged during the intended period of biomedical application. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Modelling of performance of PEM fuel cells with conventional and interdigitated flow fields

A simple mathematical model is developed to investigate the superiority of the interdigitated flow field design over the conventional one, especially in terms of maximum power density. Darcy's equation for porous media and the standard diffusion equation with effective diffusivity are used in the gas diffuser, and a coupled boundary condition given by the Butler–Volmer equation is used at the catalyst layer interface. The performance of PEM fuel cells with a conventional flow field and an interdigitated flow field is studied with other appropriate boundary conditions. The theoretical results show that the limiting current density of a fuel cell with an interdigitated flow field is about three times the current density of a fuel cell with a conventional flow field. The results also demonstrate that the interdigitated flow field design can double the maximum power density of a PEM fuel cell. The modelling results compared well with experimental data in the literature.     

四唑盐细胞活力指示剂研究进展

四唑盐类化合物是细胞生物学、细胞与分子毒理学及临床诊断领域用于测定细胞活力的生物指示剂。分别综述了:1)四唑盐及其性质;2)甲臜及其四唑盐的制备方法;3)四唑盐的发展阶段、市场需求以及商品化四唑盐基于比色技术在细胞毒性、抗菌药物的筛选及抗菌药敏实验等方面的应用,并对其发展前景进行了展望。     

The influence of collector and bacterial cell surface properties on the deposition of oral streptococci in a parallel plate flow cell

Abstraet-The deposition of two strains of oral streptococci on collector surfaces with different surface free energies has been studied in a parallel plate flow cell at various buffer concentrations. One of the strains, Streptococcus salivarius HB, was characterized by the presence of proteinaceous surface appendages with lengths between 72 and 178 nm; whereas S. salivarius HB-C12, a spontaneous mutant of S. salivarius HB, was devoid of all surface appendages. Large differences were observed between the deposition rates of the two strains which were not predicted by theoretical calculations based on the convective diffusion equation using a DLVO-type potential function. This difference was attributed to the influence of the surface appendages of S. salivarius HB, which were absent on the mutant strain S. salivarius HB-C12. Furthermore, an additional attractive interaction, not accounted for in the DLVO theory, was needed in order to match the experimental deposition rates on the low surface free energy collector surfaces with theoretical predictions.     

Optimal conditions for labelling of 3T3 fibroblasts with magnetoliposomes without affecting cellular viability

A comparative study that deals with the internalisation of different types of magnetoliposomes (MLs) by 3T3 fibroblasts revealed that cationic MLs proved to be superior to neutral and anionic ones. Internalisation was visualised both by optical light and transmission electron microscopy. The latter showed that the cationic MLs ultimately ended up in lysosomal structures. The effect of increasing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) concentrations in the cationic ML coat has been elucidated. High uptake efficiency was only achieved with MLs that carry a high DOTAP payload. However, these structures also demonstrated toxic effects. The use of the saturated distearoyl analogue (DSTAP) at identical concentrations led to improved uptake efficiency and lower toxicity. By using iron-oxide-free vesicles, it was shown that the toxicity was due to lipid bilayer constituents and not the iron oxide. In conclusion, the use of DMPC-DSTAP (96.67:3.33; molar ratio) MLs results in an extremely high labelling of 3T3 fibroblasts with iron oxides (47.66 pg Fe per cell) without evoking any influence on cell viability.     

zeta电位与菌悬液絮凝活性

通过克雷伯杆菌、有机高分子絮凝剂表面zeta电位分析和絮凝实验,初步研究了有机高分子絮凝剂对克雷伯杆菌的絮凝特性和絮凝机理。zeta电位测定表明克雷伯杆菌的等电点大致为2.2,阳离子聚丙烯酰胺（cPAM）表面的零电点约为8.0,非离子聚丙烯酰胺（nPAM）和阴离子聚丙烯酰胺（aPAM）表面均带有少量负电荷。pH<8,cPAM带正电,能与带负电的菌体电中和,显示良好的絮凝效果,最佳絮凝条件为pH7,絮凝率（FR）达94.5%。加入无机电解质后,nPAM絮凝效果变化最明显,pH4时絮凝率达95.1%,结果表明,克雷伯杆菌发酵液絮凝除菌过程是以电性中和为主,吸附架桥为辅。     

Membrane reactor immobilized with palladium‐loaded polymer nanogel for continuous‐flow Suzuki coupling reaction

A catalytic membrane reactor, which was immobilized with palladium‐loaded nanogel particles (NPs), was developed for continuous‐flow Suzuki coupling reaction. Palladium‐loaded membranes were prepared by immobilization of NPs, adsorption of palladium ions, and reduction into palladium(0). The presence of palladium in the membrane was confirmed by the scanning electron microscopy; palladium aggregation was not observed. The catalytic activity of the membrane reactor in continuous‐flow Suzuki coupling reaction was approximately double that of a comparable reactor in which palladium ions were directly adsorbed onto an aminated membrane. This was attributed to the formation of small palladium particles. The reusability in the continuous‐flow system was higher than that in a batch system, and the palladium‐loaded membrane reactor had high long‐term stability. © 2014 American Institute of Chemical Engineers AIChE J, 61: 582–589, 2015     

Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pH_i), and changes in intracellular calcium concentration ([Ca²⁺]_i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (E_m) and pH_i changes, and progesterone-induced [Ca²⁺]_i increases. Our results show that TLFC is a robust method to measure absolute E_m and pH_i values and to qualitatively evaluate [Ca²⁺]_i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pH_i alkalinization, and increased [Ca²⁺]_i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.     

Experimental study on the effect of reactant flow arrangements on the current distribution in proton exchange membrane fuel cells

Current distribution in a proton exchange membrane fuel cell (PEMFC) is significantly influenced by reactant flow configurations. In this study, the current distribution has been measured experimentally using a segmented flow-field plate and printed circuit board (PCB). Local current distributions for a PEMFC with serpentine flow field and three different flow arrangements including co-flow, cross-flow, and counter-flow arrangements for the anode and cathode streams are investigated along with the effect of flow channel orientation. It is shown that the counter-flow arrangement yields most uniform distribution for the current density, whereas the co-flow arrangement results in a considerable variation in the current density from the reactant gas stream inlet to exit. Flow channel orientation can also impact the cell performance and the current distribution appreciably. The limiting hydrogen concentration at the anode side due to the low stoichiometry condition can have a predominant effect on the current distribution and cell performance.     

Operation viability and performance of solid oxide fuel cell fuelled by different feeds

The performances of solid oxide fuel cells (SOFCs) fed by different types of feed, i.e. biogas, biogas-reformed feed, methane-reformed feed and pure hydrogen, are simulated in this work. Maximum temperature gradient and maximum cell temperature are regarded as indicators for operation viability investigation whereas power density and electrical efficiency are considered as performance indicators. The change in operating parameters, i.e. excess air, fuel feed rate and operating voltage, affects both the performance and operation viability of SOFC, and therefore, these operating parameters should be carefully selected to obtain best possible power density and reasonable temperature and temperature gradient. Pure hydrogen feed offers the highest SOFC performance among the other feeds. Extremely high excess air is required for SOFC fed by biogas to become operation viable and, in addition, its power density is much lower than those of SOFCs fed by the other feeds. Methane-reformed feed offers higher power density than biogas-reformed feed since H₂ concentration of the former one is higher.     

SEM observation of wet biological specimens pretreated with room-temperature ionic liquid

A facile pretreatment process for SEM: The use of room temperature ionic liquids (RTILs) provides an interesting method for SEM of biological specimens. We used a novel and concise method of pretreatment, excluding fixation or Au sputtering steps. Fine and smooth-textured SEM images of a wide variety of biological specimens treated in this way were observed without artefacts.     

Membranes for separation of biomacromolecules and bioparticles via flow field‐flow fractionation

Flow field‐flow fractionation (FlFFF) is a liquid‐phase separation technique that can separate macromolecules and particles on the basis of differences in their diffusion coefficient, and therefore their size. The membrane in the FlFFF channel is one of the most important elements involved in accomplishing the separation. This mini‐review focuses on requirements and important points related to membranes used in FlFFF channels. Unless it is selected properly, the membrane can be a weak point of the technique due to risks such as sample loss because of the interactions between the sample and the membrane. © 2014 Society of Chemical Industry