Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications.     

Fast on-demand droplet fusion using transient cavitation bubbles

A method for on-demand droplet fusion in a microfluidic channel is presented using the flow created from a single explosively expanding cavitation bubble. We test the technique for water-in-oil droplets, which are produced using a T-junction design in a microfluidic chip. The cavitation bubble is created with a pulsed laser beam focused into one droplet. High-speed photography of the dynamics reveals that the droplet fusion can be induced within a few tens of microseconds and is caused by the rapid thinning of the continuous phase film separating the droplets. The cavitation bubble collapses and re-condenses into the droplet. Droplet fusion is demonstrated for static and moving droplets, and for droplets of equal and unequal sizes. Furthermore, we reveal the diffusion dominated mixing flow and the transport of a single encapsulated cell into a fused droplet. This laser-based droplet fusion technique may find applications in micro-droplet based chemical synthesis and bioassays.     

Red blood cell rheology using single controlled laser-induced cavitation bubbles

The deformability of red blood cells (RBCs) is an important property that allows the cells to squeeze through small capillary vessels and can be used as an indicator for disease. We present a microfluidic based technique to quantify the deformability of RBCs by stretching a collection of RBCs on a timescale of tens of microseconds in a microfluidic chamber. This confinement constrains the motion of the cell to the imaging plane of the microscope during a transient cavitation bubble event generated with a focused and pulsed laser. We record and analyze the shape recovery of the cells with a high-speed camera and obtain a power law in time, consistent with other dynamic rheological results of RBCs. The extracted exponents are used to characterize the elastic properties of the cells. We obtain statistically significant differences of the exponents between populations of untreated RBCs and RBCs treated with two different reagents: neuraminidase reduces the cell rigidity, while wheat germ agglutinin stiffens the cell confirming previous experiments. This cavitation based technique is a candidate for high-throughput screening of elastic cell properties because many cells can be probed simultaneously in situ, thus with no pre-treatment.     

Laser-induced cavitation based micropump

Lab-on-a-chip devices are in strong demand as versatile and robust pumping techniques. Here, we present a cavitation based technique, which is able to pump a volume of 4000 microm3 within 75 micros against an estimated pressure head of 3 bar. The single cavitation event is created by focusing a laser pulse in a conventional PDMS microfluidic chip close to the channel opening. High-speed photography at 1 million frames s(-1) resolves the flow in the supply channel, pump channel, and close to the cavity. The elasticity of the material affects the overall fluid flow. Continuous pumping at repetition rates of up to 5 Hz through 6 mm long square channels of 20 microm width is shown. A parameter study reveals the key-parameters for operation: the distance between the laser focus and the channel, the maximum bubble size, and the chamber geometry.     

Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator

An understanding of chemotaxis at the level of cell-molecule interactions is important because of its relevance in cancer, immunology, and microbiology, just to name a few. This study quantifies the effects of flow on cell migration during chemotaxis in a microfluidic device. The chemotaxis gradient within the device was modeled and compared to experimental results. Chemotaxis experiments were performed using the chemokine CXCL8 under different flow rates with human HL60 promyelocytic leukemia cells expressing a transfected CXCR2 chemokine receptor. Cell trajectories were separated into x and y axis components. When the microchannel flow rates were increased, cell trajectories along the x axis were found to be significantly affected (p < 0.05). Total migration distances were not affected. These results should be considered when using similar microfluidic devices for chemotaxis studies so that flow bias can be minimized. It may be possible to use this effect to estimate the total tractile force exerted by a cell during chemotaxis, which would be particularly valuable for cells whose tractile forces are below the level of detection with standard techniques of traction-force microscopy.     

Interfacial thermocapillary vortical flow for microfluidic mixing

We present a method for the mixing of fluids in a quasi two-dimensional system with low Reynolds number by means of generating a vortical flow. A two-dimensional cavitation bubble is induced in liquid-expanded phase by locally heating a Langmuir monolayer at the air/liquid interface with an IR laser. The laser-induced cavitation bubble works as a microfluidic pump and generates a thermocapillary flow around the pump. As a result, the surrounding liquid-expanded phase flows in one direction. Perturbing the thermocapillary flow with solid folds that are created by compression and reexpansion of the monolayer induces the vortical flow behind the folds. Applying the equation of creeping flow, we find a torque halfway from the center causing the vortical flow. The vorticity created in this way stretches the liquid-expanded and gaseous phase in the azimuthal direction and at the same time thins both phases in the radial direction. If the vortical flow could be maintained long enough to reach a radial thinning that would allow the interdiffusion of surfactants at the surface, then this technique would open a route for the effective two-dimensional microfluidic mixing at low Reynolds numbers.     

Nonequilibrium bubbles in a flowing langmuir monolayer

We investigate the nonequilibrium behavior of two-dimensional gas bubbles in Langmuir monolayers. A cavitation bubble is induced in liquid expanded phase by locally heating a Langmuir monolayer with an IR-laser. At low IR-laser power the cavitation bubble is immersed in quiescent liquid expanded monolayer. At higher IR-laser power thermo capillary flow around the laser-induced cavitation bubble sets in. The thermo capillary flow is caused by a temperature dependence of the gas/liquid line tension. The slope of the line tension with temperature is determined by measuring the thermo capillary flow velocity. Thermodynamically stable satellite bubbles are generated by increasing the surface area of the monolayer. Those satellite bubbles collide with the cavitation bubble. Upon collision the satellite bubbles either coalesce with the cavitation bubble or slide past the cavitation bubble. Moreover we show that the satellite bubbles can also be produced by the emission from the laser-induced cavitation bubbles.     

Hydrophilic strips for preventing air bubble formation in a microfluidic chamber

In a microfluidic chamber, unwanted formation of air bubbles is a critical problem. Here, we present a hydrophilic strip array that prevents air bubble formation in a microfluidic chamber. The array is located on the top surface of the chamber, which has a large variation in width, and consists of a repeated arrangement of super‐ and moderately hydrophilic strips. This repeated arrangement allows a flat meniscus (i.e. liquid front) to form when various solutions consisting of a single stream or two parallel streams with different hydrophilicities move through the chamber. The flat meniscus produced by the array completely prevents the formation of bubbles. Without the array in the chamber, the meniscus shape is highly convex, and bubbles frequently form in the chamber. This hydrophilic strip array will facilitate the use of a microfluidic chamber with a large variation in width for various microfluidic applications.     

High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel

In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.     

Effect of 1.7 MHz ultrasound on a gas-vacuolate cyanobacterium and a gas-vacuole negative cyanobacterium

Ultrasonic signals propagated through medium were directly applied to unicellular cyanobacterium cell surfaces to investigate the biological effects induced by ultrasound. The gas-vacuolate cyanobacterium Microcystis aeruginosa and the gas-vacuole negative cyanobacterium Synechococcus PCC 7942 responded differently to ultrasound. When M. aeruginosa was irradiated by 1.7 MHz ultrasound at 0.6 W cm(-2) every day, it showed a decrease of nearly 65% in biomass increment, and this group's generation time increased twice as much as the control. While Synechococcus culture irradiated every day still grew as fast as the control, and its final biomass was as much as the control. The value of the electric conductivity change (Deltasigma) sharply increased in Microcystis suspension during the exposure process, which revealed more ultrasonic cavitation yield in liquid related to the gas-vacuolate cyanobacteria. The relative malondialdehyde (MDA) content, a quantitative indicator of lipid peroxidation, increased by 65% in Microcystis cells and 9% in Synechoccus cells after ultrasonic irradiation. Moreover, the membrane permeability, quantified by measuring the relative amount of electrolyte leaking out of cells, increased to more than 60% in the Microcystis cells. The results indicated that Microcystis cells were susceptible to ultrasonic stress. According to Rayleigh-Plesset's bubble activation theory, 1.7 MHz ultrasound approached the eigenfrequency of gas-vacuolate cells. The present investigation suggested the importance of the cavitational effect relative to intracellular gas-vacuoles in the loss of cell viability. In summary, 1.7 MHz ultrasonic irradiation was effective in preventing water-bloom forming cyanobacteria from growing rapidly due to changes in the functioning and integrity of cellular and subcellular structures.     

Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus

We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.     

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.     

The dynamics of a bubble ensemble in a cavitation field

A system of equations was obtained to describe the dynamics of bubbles in a cavitation cloud taking into account the interaction of pulsating bubbles involved in translational motion. The kinetics of cavitation bubble concentration changes, changes in the compressibility of the liquid, and phase transitions within a cavitation bubble and in the neighboring volume of the liquid were taken into account. The role played by bubble deformation in a cavitation cloud was considered. The Bernoulli pressure effect was shown to be negligible. The interaction of cavitation bubbles was a substantial factor that strongly influenced the dynamics of bubbles. It was suggested that there was at least one more mechanism that reduced sonoluminescence intensity from the multiple-bubble cavitation field, namely, a fairly high efficiency of sonoluminescence quenching could additionally be related to the arrival of a cumulative liquid stream at the central cavitation bubble region, where the concentration of active species was high. The dynamics of bubbles in the cavitation field is not only related to the expansion and compression of cavitation bubbles in the acoustic field, but also governed to a great extent by their interaction, translational motion, deformation, and the influence of cumulative streams penetrating the bubbles.     

An electron diffusion model of the space-time distribution of radicals in a multibubble cavitation field

Conditions under which electric discharges can arise in cavitation bubbles pulsating in a multibubble cavitation field are considered. Possible electric discharge types are discussed. It is shown that, in an electric breakdown in a cavitation bubble, the probability of the formation of a streamer (all the more, a leader) or corona discharge is negligibly small. It was found that, in a cavitation bubble, the development of an electron avalanche is most probable. The basic parameters of an elementary electron avalanche are estimated.     

Electrochemiluminescence analysis of folate receptors on cell membrane with on-chip bipolar electrode

In this paper we report a transparent bipolar electrode based microfluidic chip-electrochemiluminescence (ECL) system for sensitive detection of folate receptors (FR) on cell membranes. This integrated system consists of a poly(dimethylsiloxane) (PDMS) layer containing a microchannel and a glass bottom sheet with indium tin oxide (ITO) strips as bipolar detectors. The ITO strips are fabricated using a PDMS micromold with carbon ink as a protective layer in place of traditional photoresist. The configuration of the bipolar electrode has great influence on the ECL intensity of Ru(bpy)(3)(2+)/tripropylamine(TPA) system. Further studies show that folic acid (FA) can strongly inhibit the ECL of the Ru(bpy)(3)(2+)/TPA system. Based on specific recognition between FA and FR on cell membrane, this microfluidic chip-ECL system is successfully applied for detecting the level of FR on human cervical tumor (HL-60) cells and MEF cells. It is found that the ECL intensity increases with the number of HL-60 cells in the range of 21 to 3.28 × 10(4) cells/mL. The average level of FR on HL-60 cells is calculated to be 8.05 ± 0.75 × 10(-18) mol/cell. While for MEF cells, it shows a much slower ECL increment than HL-60 cells due to the much lower FR level on MEF cells (5.30 ± 0.61 × 10(-19) mol/cell). Moreover, exocytosis of FA after FR mediated endocytosis was observed according to the change of the ECL signal with the incubation time of HL-60 cells in the FA- Ru(bpy)(3)(2+)/TPA system.     

The enhanced diffusional mixing for latex immunoagglutination assay in a microfluidic device

Latex immunoagglutination assay in a microfluidic device is expected to be even easier than its large-sized, commercialized counterpart. However, such demonstration has had a limited success due to the difficulties in mixing in a microfluidic device, especially for the microparticles used in latex immunoagglutination assay. The primary goal of this work is to improve diffusional mixing towards the successful latex immunoagglutination in a microfluidic devices without any non-specific binding. To this end, SDS (sodium dodecyl sulfate, an ionic surfactant) or Tween 80 (polyethylene sorbitol ester, a non-ionic surfactant) was added to the antibody-conjugated polystyrene (PS) microparticle suspension. These surfactant-added particle suspensions were mixed with the target antigen solution at the Y-junction of a microfluidic device. The immunoagglutination and the diffusion behavior were visually identified with an inverted light microscope. Both surfactants showed some problems such as non-specific binding (with SDS) or very poor diffusion (with Tween 80). As an alternative approach, therefore, highly carboxylated PS microparticles, where the surface is saturated with carboxyl-terminated side chains, were evaluated without using any surfactants. These particles showed very low non-specific binding comparable to that with Tween 80 and good diffusional mixing equivalent to that with SDS.     

Dynamic adsorption properties of n-alkyl glucopyranosides determine their ability to inhibit cytolysis mediated by acoustic cavitation

Suspensions of human leukemia (HL-60) cells readily undergo cytolysis when exposed to ultrasound above the acoustic cavitation threshold. However, n-alkyl glucopyranosides (hexyl, heptyl, and octyl) completely inhibit ultrasound-induced (1057 kHz) cytolysis (Sostaric, et al. Free Radical Biol. Med. 2005, 39, 1539-1548). The efficacy of protection from ultrasound-induced cytolysis was determined by the n-alkyl chain length of the glucopyranosides, indicating that protection efficacy depended on adsorption of n-alkyl glucopyranosides to the gas/solution interface of cavitation bubbles and/or the lipid membrane of cells. The current study tests the hypothesis that "sonoprotection" (i.e., protection of cells from ultrasound-induced cytolysis) in vitro depends on the adsorption of glucopyranosides at the gas/solution interface of cavitation bubbles. To test this hypothesis, the effect of ultrasound frequency (from 42 kHz to 1 MHz) on the ability of a homologous series of n-alkyl glucopyranosides to protect cells from ultrasound-induced cytolysis was investigated. It is expected that ultrasound frequency will affect sonoprotection ability since the nature of the cavitation bubble field will change. This will affect the relative importance of the possible mechanisms for ultrasound-induced cytolysis. Additionally, ultrasound frequency will affect the lifetime and rate of change of the surface area of cavitation bubbles, hence the dynamically controlled adsorption of glucopyranosides to their surface. The data support the hypothesis that sonoprotection efficiency depends on the ability of glucopyranosides to adsorb at the gas/solution interface of cavitation bubbles.     

A hydrogel-based microfluidic device for the studies of directed cell migration

We have developed a hydrogel-based microfluidic device that is capable of generating a steady and long term linear chemical concentration gradient with no through flow in a microfluidic channel. Using this device, we successfully monitored the chemotactic responses of wildtype Escherichia coli (suspension cells) to alpha-methyl-DL-aspartate (attractant) and differentiated HL-60 cells (a human neutrophil-like cell line that is adherent) to formyl-Met-Leu-Phe (f-MLP, attractant). This device advances the current state of the art in microchemotaxis devices in that (1) it demonstrates the validity of using hydrogels as the building material for a microchemotaxis device; (2) it demonstrates the potential of the hydrogel based microfluidic device in biological experiments since most of the proteins and nutrients essential for cell survival are readily diffusible in hydrogel; (3) it is capable of applying chemical stimuli independently of mechanical stimuli; (4) it is straightforward to make, and requires very basic tools that are commonly available in biological labs. This device will also be useful in controlling the chemical and mechanical environment during the formation of tissue engineered constructs.     

Chromatographic identification of benzene hydrogenation products in the preparation of artificial diamonds using a symmetric cavitation bubble collapse

The process of symmetric cavitation bubble collapse, which had been only theoretically discussed previously, was performed for the first time. The formation of diamond matter in an adiabatic cavitation bubble collapse in a carbon-containing liquid (benzene) was demonstrated. Benzene hydrogenation products—formed in the course of the formation of diamond matter under conditions of a symmetric adiabatic cavitation bubble collapse as the result of a high-energy explosive action on a hydrocarbon working fluid (benzene)—were identified for the first time using gas chromatography.     

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.