Microfluidic flow focusing: drop size and scaling in pressure versus flow-rate-driven pumping

We experimentally study the production of micrometer-sized droplets using microfluidic technology and a flow-focusing geometry. Two distinct methods of flow control are compared: (i) control of the flow rates of the two phases and (ii) control of the inlet pressures of the two phases. In each type of experiment, the drop size l, velocity U and production frequency f are measured and compared as either functions of the flow-rate ratio or the inlet pressure ratio. The minimum drop size in each experiment is on the order of the flow focusing contraction width a. The variation in drop size as the flow control parameters are varied is significantly different between the flow-rate and inlet pressure controlled experiments.     

Counting of Escherichia coli by a microflow cytometer based on a photonic–microfluidic integrated device

Counting of Escherichia coli DH5α‐cell suspensions in PBS is performed using a microflow cytometer based on a photonic–microfluidic integrated device. Side‐scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a hemocytometer. The detection efficiency is correlated to the ratio of sample to sheath flow rates. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2–4 μm) as their scattering intensities are different.     

The effects of flow type on aptamer capture in differential mobility cytometry cell separations

In this work, differential mobility cytometry (DMC) was used to monitor cell separation based on aptamer recognition for target cells. In this device, open-tubular capillaries coated with Sgc8 aptamers were used as affinity chromatography columns for separation. After cells were injected into the columns, oscillating flow was generated to allow for long-term cell adhesion studies. This process was monitored by optical microscopy, and differential imaging was used to analyze the cells as they adhered to the affinity surface. We investigated the capture time, capture efficiency, purity of target and control cells, as well as the reusability of the affinity columns. Capture time for both CCRF-CEM cells and Jurkat T cells was 0.4 ± 0.2 s, which demonstrated the high separation affinity between aptamers and target cells. The capture efficiency for CCRF-CEM cells was 95% and purity was 99% in a cell mixture. With the advantage of both high cell capture efficiency and purity, DMC combined with aptamer-based separation emerges as a powerful tool for rare cell enrichment. In addition, aptamer-based DMC channels were found to be more robust than antibody based channels with respect to reuse of the separation device.     

The good, the bad and the twisted: a survey of ligand geometry in protein crystal structures

The protein databank now contains the structures of over 11,000 ligands bound to proteins. These structures are invaluable in applied areas such as structure-based drug design, but are also the substrate for understanding the energetics of intermolecular interactions with proteins. Despite their obvious importance, the careful analysis of ligands bound to protein structures lags behind the analysis of the protein structures themselves. We present an analysis of the geometry of ligands bound to proteins and highlight the role of small molecule crystal structures in enabling molecular modellers to critically evaluate a ligand model’s quality and investigate protein-induced strain.     

Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry

The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.     

微流控芯片用于流式细胞术的基础研究

自行设计并加工了玻璃微流控芯片,并将其与流式细胞术相结合,采用羟丙基甲基纤维素(HPMC)的磷酸盐溶液为缓冲体系和鞘液,解决了微粒在微芯片中流动的若干问题,使其状态可以得到更有效的控制.采用自行组装的激光诱导荧光装置并结合动电聚焦技术,实现了对荧光微球的计数,并可通过荧光倒置显微镜实时观察到微通道内微球的实际流动情况.方法简单,操作方便,并且具有仪器体积小、试剂及样品用量少和分析速度快等优点.     

Antimicrobial properties of biosynthesized silver nanoparticles studied by flow cytometry and related techniques

This work reports the effect of silver bionanoparticles (Bio(AgNPs) synthesized by Actinobacteria CGG 11n on selected Gram (+) and Gram (–) bacteria. Flow cytometry, classical antibiogram method and fluorescent microscopy approach was used for evaluation of antimicrobial activity of Bio(AgNPs) and their combination with antibiotics. Furthermore, the performed research specified the capacity of flow cytometry method as an alternative to the standard ones and as a complementary method to electromigration techniques. The study showed antibacterial activity of both BioAgNPs and the combination of antibiotics/BioAgNPs against all the tested bacteria strains in comparison with a diffusion, dilution and bioautographic methods. The synergistic effect of antibiotics/BioAgNPs combination (e.g. kanamycin, ampicillin, neomycin and streptomycin) was found to be more notable against Pseudomonas aeruginosa representing a prototype of multi‐drug resistant “superbugs” for which effective therapeutic options are very limited.     

The use of immortalized cell lines in GPCR screening: the good, bad and ugly

For most membrane-bound molecular targets, including G protein linked receptors (GPCRs), the optimal approach in drug discovery involves the use of cell based high throughput screening (HTS) technologies to identify compounds that modulate target activity. Most GPCRs have been cloned and can therefore be routinely expressed in immortalized cell lines. These cells can be easily and rapidly grown in unlimited quantities making them ideal for use in current HTS technologies. A significant advantage of this approach is that immortalized recombinant cells provide a homogenous background for expression of the target which greatly facilitates consistency in screening, thus allowing for a better understanding of the mechanism of action of the interacting compound or drug. Nonetheless, it is now evident that numerous disparities exist between the physiological environment of screening systems using recombinant cells and natural tissues. This has lead to a problem in the validity of the pharmacological data obtained using immortalized cells in as much as such cells do not always reflect the desired clinical efficacy and safety of the compounds under examination. This brief review discusses these issues and describes how they influence the discovery of drugs using modern HTS.     

Detection of unlabeled particles in the low micrometer size range using light scattering and hydrodynamic 3D focusing in a microfluidic system

In this paper, we describe a microfluidic device composed of integrated microoptical elements and a two-layer microchannel structure for highly sensitive light scattering detection of micro/submicrometer-sized particles. In the two-layer microfluidic system, a sample flow stream is first constrained in the out-of-plane direction into a narrow sheet, and then focused in-plane into a small core region, obtaining on-chip three-dimensional (3D) hydrodynamic focusing. All the microoptical elements, including waveguides, microlens, and fiber-to-waveguide couplers, and the in-plane focusing channels are fabricated in one SU-8 layer by standard photolithography. The channels for out-of-plane focusing are made in a polydimethylsiloxane (PDMS) layer by a single cast using a SU-8 master. Numerical and experimental results indicate that the device can realize 3D hydrodynamic focusing reliably over a wide range of Reynolds numbers (0.5 < Re < 20). Polystyrene particles of three sizes (2, 1, and 0.5 μm) were measured in the microfluidic device with integrated optics, demonstrating the feasibility of this approach to detect particles in the low micrometer size range by light scattering detection.     

Electroporation of cells in microfluidic devices: a review

In recent years, several publications on microfluidic devices have focused on the process of electroporation, which results in the poration of the biological cell membrane. The devices involved are designed for cell analysis, transfection or pasteurization. The high electric field strengths needed are induced by placing the electrodes in close proximity or by creating a constriction between the electrodes, which focuses the electric field. Detection is usually achieved through fluorescent labeling or by measuring impedance. So far, most of these devices have only concerned themselves solely with the electroporation process, but integration with separation and detection processes is expected in the near future. In particular, single-cell content analysis is expected to add further value to the concept of the microfluidic chip. Furthermore, if advanced pulse schemes are employed, such microdevices can also enhance research into intracellular electroporation.     

Revisit of wall‐induced lateral migration in particle electrophoresis through a straight rectangular microchannel: Effects of particle zeta potential

Previous studies have reported a lateral migration in particle electrophoresis through a straight rectangular microchannel. This phenomenon arises from the inherent wall‐induced electrical lift that can be exploited to focus and separate particles for microfluidic applications. Such a dielectrophoretic‐like force has been recently found to vary with the buffer concentration. We demonstrate in this work that the particle zeta potential also has a significant effect on the wall‐induced electrical lift. We perform an experimental study of the lateral migration of equal‐sized polystyrene particles with varying surface charges under identical electrokinetic flow conditions. Surprisingly, an enhanced focusing is observed for particles with a faster electrokinetic motion, which indicates a substantially larger electrical lift for particles with a smaller zeta potential. We speculate this phenomenon may be correlated with the particle surface conduction that is a strong function of particle and fluid properties.     

The Integrated Calibration Method: Comparison of Various Flow Approaches

The integrated calibration method (ICM) is a novel calibration approach merging interpolative and extrapolative modes in a single procedure. Thanks to this strategy, chemical analysis can be performed along with a diagnosis of systematic calibration errors that leads to better accuracy of analytical results. The paper describes how the ICM can be adapted to different flow techniques: continuous flow, flow-injection, and sequential-injection ones. Instrumental systems dedicated to each flow technique have been presented and their operation has been explained. The systems were used for UV/VIS chromium determination in water samples and compared with each other in terms of precision and accuracy of the obtained results along with time and reagent consumption.     

The use of a micropump based on capillary and evaporation effects in a microfluidic flow injection chemiluminescence system

The performance of a micropump operating on evaporation and capillary effects, developed for microfluidic (lab-on-a-chip) systems, was studied employing it as the fluid drive in a microfluidic flow injection (FI) system, with chemiluminescence (CL) detection. The micropump featured simple structure, small dimensions, low fabrication cost and stable and adjustable flow-rates during long working periods. Using a micropump with 6.6 cm² evaporation area, with the ambient temperature and relative humidity fluctuating within 2 h in the ranges 20-21 °C and 30-32%, respectively, an average flow-rate of 3.02 μL/min was obtained, with a precision better than 1.2% R.S.D. (n = 61). When applied to the microchip FI-CL system using the luminol/hexacyanoferrate/H₂O₂ reaction, a precision of 1.4% R.S.D. (n = 11) was obtained for luminol at a sampling frequency of 30 h⁻¹.     

Real-time monitoring of cell migration,phagocytosis and cell surface receptor dynamics using a novel,live-cell opto-microfluidic technique

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic “Lab-in-a-Trench” (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell–cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective–diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.     

Investigation of bacterial viability from incubated saliva by application of flow cytometry and hyphenated separation techniques

The aim of the study was determination of bacterial viability in saliva samples and finding a correlation between microbiological and volatile profiles of saliva depending on incubation time. Bacteria colonizing healthy oral cavities were also identified. Twelve healthy adults donated unstimulated saliva samples. Flow cytometry, optical density measurements and colony‐forming unit (CFU) counting method were employed for analyses of native and inoculated saliva after 0, 1, 2, 24, and 48 h of incubation. Volatile profiles were acquired using headspace‐solid phase microextraction‐gas chromatography/mass spectrometry (HS‐SPME‐GC/MS). Oral bacteria were the most viable within 2 h after collection of saliva. Extension of incubation time to 48 h caused considerable decrease in live bacteria counts and sharp increase in dead bacteria counts. The most prevalent strain was Sphingomonas paucimobilis (26.67%). The number of volatiles raised from 5 to 27 with incubation time and most of them were putrefaction products, such as methanethiol, indole and pyrrole. HS‐SPME‐GC/MS method is insufficient for volatile profiling of “fresh” saliva and should be directed rather to investigation of bacterial metabolites.     

Optical trapping in micro- and nanoconfinement systems: Role of thermo-fluid dynamics and applications

In this mini-review, recent advances on the role of a focused laser in micro- and nanofluidic systems is widely introduced with special interest in thermo-fluid dynamical aspects and their importance in optical manipulation. As a brief introduction to microfluidic systems, we describe the advantages and challenges of the use of micro- and nanoscale confinement in optical trapping, as well as standard fabrication techniques for micro- and nanofluidic systems. From thermo-fluid dynamical viewpoints, various phenomena that accompany a laser irradiation to fluidic devices, are explained in detail. These phenomena can affect the optical trapping of target materials significantly, and are classified into two categories: one that induces the fluid flow around the target and another that directly acts on it as an external force. These classes are reviewed by shedding light on some recent cutting-edge researches for optical manipulation. Some applications using thermo-fluid dynamics in microfluidic systems for the measurement of optical forces and for the separation, measurement, and detection of target materials are also introduced.     

Evaluation of transversal and longitudinal dispersion in a flow injection system by exploiting laser induced fluorescence: influence of flow-cell positioning

A fully mechanized set-up was built for the experimental determination of bi-dimensional dispersion with high spatial resolution (2400 μm²). Gravitational and wall effects in a single stream were evaluated by using time-based sampling and a micro-flow cell. Vertical upward and downward flows as well as horizontal flows were investigated. Ethylene glycol (MEG) and Rhodamine B in MEG were used as carrier and sample solutions, respectively. Longitudinal profiles were obtained by laser induced total fluorescence (LIF) at up to 19 transversal sites and combined to generate high-resolution bi-dimensional profiles. A two frontal maxima pattern was observed for all flows. The volumetric fraction of RB shape was highly stretched for downward flow and there was high asymmetry for horizontal flow. The sensitivity of three dispersion parameters was evaluated: maximum peak value, peak half-width at half-height, and peak area.Data modeling showed that the tanks-in-series was more sensitive to wall effects, had good adjustment with only one tank for upward and horizontal flow and needed two tanks for downward flow which was attributed to the latter having higher dispersion. A black box empirical modeling described better the gravitational effect and allowed to identify a parameter sensitive to upward and downward flow as well as hinting to two inner streams within the horizontal flow. It also pointed to a wall dispersion contribution of twice that of the liquid-liquid dispersion.     

Flow injection determinations of citric acid: a review

A review of the flow injection (FI) analytical methods used to determine citric acid (CA) is presented. These flow determinations are described and compared according to the detection technique used. In addition to the advantages involving the use of the continuous flow systems, the analytical figures of merit and interferences are also discussed.     

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials,magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.     

Two-photon fluorescence excitation using an integrated optical microcavity: a promising tool for biosensing of natural chromophores

Application of an integrated optics (IO) microcavity (MC) for evanescent excitation of two-photon excited fluorescence (TPF) is demonstrated. The MC provides a high local intensity, which is required for the TPF, because of resonant enhancement of the intracavity power and a strong two-dimensional confinement of the guided mode. Numerical estimations show a large increase, by more than a factor of 10⁴ of the TPF intensity at the MC compared to a conventional straight waveguide. This will lead to a significant improvement of the detection limits of UV-absorbing chromophores (down to 10⁻⁸ M) when using the MC as a biosensor. Feasibility of TPF excitation using an IO MC is confirmed experimentally for the first time.