Optical tweezers applied to a microfluidic system

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.     

Holographic optical tweezers and their relevance to lab on chip devices

During the last decade, optical tweezers have been transformed by the combined availability of spatial light modulators and the speed of low-cost computing to drive them. Holographic optical tweezers can trap and move many objects simultaneously and their compatibility with other optical techniques, particularly microscopy, means that they are highly appropriate to lab-on-chip systems to enable optical manipulation, actuation and sensing.     

A microfluidic method to study demulsification kinetics

We present the results of experiments studying droplet coalescence in a dense layer of emulsion droplets using microfluidic circuits. The microfluidic structure allows direct observation of collisions and coalescence events between oil droplets dispersed in water. The coalescence rate of a flowing hexadecane-in-water emulsion was measured as a function of the droplet velocity and droplet concentration from image sequences measured with a high-speed camera. A trajectory analysis of colliding droplet pairs allows evaluation of the film drainage profile and coalescence time t(c.) The coalescence times obtained for thousands of droplet pairs enable us to calculate coalescence time distributions for each set of experimental parameters, which are the mean droplet approach velocity (v(0)), the mean dispersed phase fraction (φ) and the mean hydraulic diameter of a droplet pair (d(p)). The expected value E(t(c)) of the coalescence time distributions scales as E(t(c)) is proportional to (v(0))(-0.105±0.043)(d(p))(0.562±0.287), but is independent of φ. We discuss the potential of the procedure for the prediction of emulsion stability in industrial applications.     

A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells

The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria.     

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen–host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.     

A microfluidic chip based liquid-liquid extraction system with microporous membrane

A robust and simple approach for microfabricated chip based liquid-liquid extraction was developed for on-chip sample pretreatment. The chip based extraction system was composed of two microfabricated glass plates with a microporous membrane sandwiched in between. A simple bonding approach using epoxy was used to achieve bonding and sealing of the L-L extraction chip. Gravity was employed to drive the aqueous and organic flows through separate channels in the extraction system, separated by the membrane. During extraction, the analyte in an aqueous sample stream was transferred through the membrane into the organic stream. The fluorescence intensity of the analyte extracted into the organic stream was monitored in situ by a laser induced fluorescence detection system. The performance of the system was demonstrated using an aqueous solution of butyl rhodamine B (BRB) and isobutanol as sample and extractant, respectively. The system proved to be an efficient means for achieving chip based microporous membrane liquid-liquid extraction. The precision of fluorescence measurements was 1.5% R.S.D. (n = 4). A linear response range of 1 × 10⁻⁷ to 1 × 10⁻⁴ M BRB was obtained with a regression equation: I = 8.00 × 10⁶ C + 4.91. An enrichment factor of ca. 3 was obtained with an extraction efficiency of 69%.     

A high-throughput microfluidic assay to study neurite response to growth factor gradients

Studying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 μg mL(-1)) and brain pulp (0.1 μL mL(-1)) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng mL(-1)) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient was measured in the presence of brain pulp and netrin-1 (1 μg mL(-1)), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p < 0.001). Slit-2 exhibited strong chemorepellant characteristics on both hippocampal and DRG neurite guidance at 250 ng mL(-1) concentration. Slit-2 also showed similar behavior on DRG neuron invasion into 3D collagen gel (p < 0.01 relative to control cultures). Taken together, the results suggest the utility of this microfluidic device to generate stable chemogradients for studying neurobiology, cell migration and proliferation, matrix remodeling and co-cultures with other cell lines, with potential applications in cancer biology, tissue engineering and regenerative medicine.     

Surface forces and drag coefficients of microspheres near a plane surface measured with optical tweezers

Optical tweezers are widely used to measure molecular forces in biology. Such measurements are often influenced by a nearby surface that can perturb both the calibration of the tweezers as well as the hydrodynamic forces acting on microspheres to which the biomolecules are attached. In this study, we have used a very stable optical tweezers setup employing a recently developed calibration method (Toli?-N?rrelykke, S. F.; Sch?ffer, E.; Howard, J.; Pavone, F. S.; Jülicher, F.; Flyvbjerg, H. Rev. Sci. Instrum. 2006, 77 (10), 103101) to determine how the calibration of the tweezers and the forces on the microspheres depend on the height above the surface. We show that the displacement sensitivity of the tweezers is modulated by a standing light wave between the microsphere and the surface. We measured the dependence of the drag coefficient on height and compared it to exact and closed-form solutions to the Navier-Stokes equations. Also, we measured the surface force gradients in different salt solutions and for different surface blocking methods. For a given blocking method, our data suggest that microspheres can experience attractive and/or repulsive forces close to surfaces. For example, a Teflon layer reduces attractive interactions, and the presence of casein can lead to long-range repulsive interactions. These measurements are a prerequisite for the accurate measurement of normal forces with respect to an interface that occur in biological molecules held between surfaces.     

Single particle analysis using fluidic, optical and electrophoretic force balance in a microfluidic system

A unique microfluidic system is developed which enables the interrogation of a single particle by using multiple force balances from a combination of optical force, hydrodynamic drag force, and electrophoretic force. Two types of polystyrene (PS) particles with almost identical size and refractive index (plain polystyrene (PS) particle - mean diameter: 2.06 μm, refractive index: 1.59; carboxylated polystyrene (PS-COOH) particles - mean diameter: 2.07 μm, refractive index: 1.60), which could not be distinguished by optical chromatography, reveal different electrokinetic behaviors resulting from the difference in their surface charge densities. The PS-COOH particles, despite their higher surface charge density when compared to the PS particles, experience a lower electrophoretic force, regardless of ionic strength. This phenomenon can be understood when the more prominent polarization of the counter ion cloud surrounding the PS-COOH particles is considered. The surface roughness of the carboxylated particles also plays an important role in the observed electrokinetic behavior.     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

A microfluidic system for high-speed reproducible DNA sizing and quantitation

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.     

Fabrication of poly(dimethylsiloxane) microfluidic system based on masters directly printed with an office laser printer

Applications of poly(dimethylsiloxane) (PDMS)-based microfluidic systems are more popular nowadays. Previous fabrication methods of the masters for PDMS microchannels require complicated steps and/or special device. In this paper, we demonstrated that the toner printed on the transparency film with the office laser printer (1200 dpi) can be used as the positive relief of the masters. The transparency film was printed in two steps in order to obtain the same printing quality for the crossed lines. With the laser-printed master, the depth of the fabricated PDMS microchannels was ca. 10 microm and the smallest width was ca. 60 microm. Surface characteristics of the PDMS/PDMS microchannels were performed with SEM. Their electrokinetic properties were investigated by the aids of the measurement of electroosmotic flow (EOF) and the Ohm's curve. Using the PDMS/PDMS microchip CE systems, electroactive biological molecules and non-electroactive inorganic ions were well separated, respectively. This simple approach could make it easy to carry out the studies of PDMS microfluidic systems in more general labs without special devices.     

PDMS 2D optical lens integrated with microfluidic channels: principle and characterization

In this paper, the fabrication and characterization of PDMS 2D-optical lenses are reported. These lenses are designed in order to improve the performance of fluorescent spectroscopy detection performed on a portable chip using optical fibers. The fabrication process of the PDMS layer is first detailed, and the patterns are then checked with a SEM. By comparing various interfacial structures, it is shown that the beam properties of the light coming out from the fiber can be modified depending on the lens curvature radius. As a consequence, for a constant dye concentration, the use of such lenses can increase the intensity of fluorescent response close to the fiber or far from the fiber, compared to the same design with a flat interface. This excitation improvement corresponding to a stronger response from the dye then consequently leads to around three times higher sensitivity of the on-chip detection method for fluorescent spectroscopy.     

A microfluidic device that forms and redirects pheromone gradients to study chemotropism in yeast

Chemotropism, or directed cell growth in response to a chemical gradient, is integral to many biological processes. The mating response of the budding yeast, Saccharomyces cerevisiae, is a well studied model chemotropic system. Yeast cells of opposite mating type signal their positions by secreting soluble mating pheromones. The mutual exchange of pheromones induces the cells to grow towards one another, resulting in mating projections or "shmoos." Yeast cells exhibit a remarkable ability to orient their growth toward the nearest potential mating partner, and to reorient (i.e., bend their mating projections) in response to a change in the direction of the pheromone gradient. Although a number of microfluidic devices have been used to generate linear pheromone gradients and to measure initial orientation, none of them have the capability to change the direction of the gradient, other than to invert it. We have developed a microfluidic device that can produce stable pheromone gradients and rapidly rotate them in 90° increments, mimicking the dynamic gradients yeast are exposed to in situ, and allowing for the study of reorientation as well as initial orientation. The mean angle of orientation exhibited by gradient-stimulated yeast cells in this device was 56.9°. In control experiments, cells subjected to pheromone coming from all four directions showed no evidence of orientation. Switching the direction of the pheromone source by 90° induced 83.6% of the polarized cells to change their direction of growth. Of these, 85.2% bent their mating projections toward the second source, demonstrating the utility of this device in the study of reorientation with specifically controlled gradients.     

A microfluidic system incorporated with peptide/Pd nanowires for heterogeneous catalytic reactions

Highly stable diphenylalanine peptide nanowires were selectively self-assembled in the reaction zone of a microfluidic system and applied for microchemical hydrogenation and Suzuki coupling reactions through the hybridization of Pd nanoparticles.     

A microfluidic chip based sequential injection system with trapped droplet liquid-liquid extraction and chemiluminescence detection

A microfluidic chip-based sequential injection system with trapped droplet liquid-liquid extraction preconcentration and chemiluminescence detection was developed for achieving high sensitivity with low reagent and sample consumption. The microfabricated glass lab-chip had a 35 mm long extraction channel, with 134 shrunken opening rectangular recesses (L 100 microm x W 50 microm x D 25 microm) arrayed within a 1 mm length on both sides of the middle section of the channel. Ketonic peroxyoxalate ester solution was filled in the recesses forming organic droplets, and keeping the aqueous sample solution flowing continuously in the extraction channel; analytes were transferred from the aqueous phase into the droplets through molecular diffusion. After liquid-liquid extraction preconcentration, catalyst and hydrogen peroxide solutions were introduced into the channel, and mixed with analytes and peroxyoxalate ester to emit chemiluminescence light. The performance of the system was tested using butyl rhodamine B, yielding a precision of 4% RSD (n = 5) and a detection limit of 10(-9) M. Within a 17 min analytical cycle, the consumptions of sample and peroxyoxalate solutions were 2.7 microL and 160 nL, respectively.     

A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis

Single-cell measurements allow a unique glimpse into cell-to-cell heterogeneity; even small changes in selected cells can have a profound impact on an organism's physiology. Here an integrated approach to single-cell chemical sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydroxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10(-14) mol to 10(-16) mol, an order of magnitude higher than observed in untreated pinealocytes.     

Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.     

Polyelectrolyte multilayers generated in a microfluidic device with pH gradients direct adhesion and movement of cells

In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three different pH values of 5, 7 and 9. Water contact angle and quartz microbalance measurements show that resulting multilayers differ in terms of wetting behaviour, layer mass and mechanical properties. The multilayer is then formed within a gradient generation microfluidic (μFL) device. Polyethylene imine or heparin solutions of pH 5 are introduced into one inlet and the same solutions but at pH 9 into another inlet of the μFL device. The pH gradient established during the multilayer formation can be visualized inside the microchamber by pH sensitive fluorophores and confocal laser scanning microscopy. From this setup it is expected that properties of multilayers displayed at distinct pH values can be realised in a gradient manner inside the μFL device. Behaviour of the osteoblast cell line MG-63 seeded and cultured on top of multilayers created inside the μFL device support this hypothesis. It is observed that more cells adhere and spread on multilayers build-up at the basic side of the μFL channel, while those cells on top of multilayers built at pH 5 are fewer and smaller. These results are consistent with the behaviour of MG-63 cells seeded on multilayers formed at discrete pH values. It is particularly interesting to see that cells start to migrate from multilayers built at pH 5 to those built at pH 9 during 6 h of culture. Overall, the presented multilayer formation setup applying pH gradients leads to surfaces that promote migration of cells.