Polycaprolactone solution–based ink for designing microfluidic channels on paper via 3D printing platform for biosensing application

This work reports a novel fabrication technique for development of channels on paper‐based microfluidic devices using the syringe module of a 3D printing syringe–based system. In this study, printing using polycaprolactone (PCL)‐based ink (Mw 70 000‐90 000) was employed for the generation of functional hydrophobic barriers on Whatman qualitative filter paper grade 1 (approximate thickness of 180 μm and pore diameter of 11 μm), which would effectively channelize fluid flow to multiple assay zones dedicated for different analyte detection on a microfluidic paper‐based analytical device (μPAD). The standardization studies reveal that a functional hydrophilic channel for sample conduction fabricated using the reported technique can be as narrow as 460.7 ± 20 μm and a functional hydrophobic barrier can be of any width with a lower limit of about 982.2 ± 142.75 μm when a minimum number of two layers of the ink is extruded onto paper. A comparison with the hydrodynamic model established for writing with ink is used to explain the width of the line printed by this system. A fluid flow analysis through a single channel system was also carried out to establish its conformity with the Washburn model, which governs the fluid flow in two‐dimensional μPAD. The presented fabrication technique proves to be a robust strategy that effectively taps the advantages of this 3D printing technique in the production of μPADs with enhanced speed and reproducibility.     

Generation and characterization of air micro-bubbles in highly hydrophobic capillaries

The generation of air microbubbles in microfluidic systems or in capillaries could be of great interest for transportation (single cell analysis, organite transportation) or for liquid compartmentation. The physicochemical characterization of air bubbles and a better understanding of the process leading to bubble generation during electrophoresis is also interesting in a theoretical point of view. In this work, the generation of microbubbles on hydrophobic Glaco™ coated capillaries has been studied in water-based electrolyte. Air bubbles were generated at the detection window and the required experimental parameters for microbubbles generation have been identified. Generated bubbles migrated against the electroosmotic flow, as would do strongly negatively charged solutes, under constant electric field. They have been characterized in terms of dimensions, electrophoretic mobility, and apparent charge.     

Mixing enhancement for high viscous fluids in a microfluidic chamber

Due to small channel dimensions and laminar flows, mixing in microfluidic systems is always a challenging task, especially for high viscous fluids. Here we report a method of enhancing microfluidic mixing for high viscous fluids using acoustically induced bubbles. The bubbles can be generated in an acoustically profiled microfluidic structure by using a piezoelectric disk activated at a working frequency range between 1.5 kHz and 2 kHz. The mixing enhancement is achieved through interactions between the oscillating bubbles and fluids. Both experimental studies and numerical simulations are conducted. In the experiments, DI water-glycerol mixture solutions with various viscosities were used. The results, based on the mixing efficiency calculated from experimentally acquired fluorescent images, showed that good mixing can occur in the DI water-glycerol solutions with their maximum viscosity up to 44.75 mPa s, which to our best knowledge is the highest viscosity of fluids in microfluidic mixing experiments. To explain the mechanisms of bubble generation, the numerical simulation results show that, corresponding to the actuations at the working frequency range used in the experiment, there exists a low pressure region where the pressure is lower than the water vapor pressure in the DI water-glycerol solutions, resulting in the generation of bubbles.     

An optimal design method for preventing air bubbles in high-temperature microfluidic devices

DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (μ-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.     

Dissolution of carbon dioxide bubbles and microfluidic multiphase flows

We experimentally study the dissolution of carbon dioxide bubbles into common liquids (water, ethanol, and methanol) using microfluidic devices. Elongated bubbles are individually produced using a hydrodynamic focusing section into a compact microchannel. The initial bubble size is determined based on the fluid volumetric flow rates of injection and the channel geometry. By contrast, the bubble dissolution rate is found to depend on the inlet gas pressure and the fluid pair composition. For short periods of time after the fluids initial contact, the bubble length decreases linearly with time. We show that the initial rate of bubble shrinkage is proportional to the ratio of the diffusion coefficient and the Henry's law constant associated with each fluid pair. Our study shows the possibility to rapidly impregnate liquids with CO(2) over short distances using microfluidic technology.     

Bubbles no more: in-plane trapping and removal of bubbles in microfluidic devices

Gas bubbles present a frequent challenge to the on-chip investigation and culture of biological cells and small organs. The presence of a single bubble can adversely impair biological function and often viability as it increases the wall shear stress in a liquid-perfused microchannel by at least one order of magnitude. We present a microfluidic strategy for in-plane trapping and removal of gas bubbles with volumes of 0.1-500 nL. The presented bubble trap is compatible with single-layer soft lithography and requires a footprint of less than ten square millimetres. Nitrogen bubbles were consistently removed at a rate of 0.14 μL min(-1). Experiments were complemented with analytical and numerical models to comprehensively characterize bubble removal for liquids with different wetting behaviour. Consistent long-term operation of the bubble trap was demonstrated by removing approximately 4000 bubbles during one day. In a case study, we successfully applied the bubble trap to the on-chip investigation of intact small blood vessels. Scalability of the design was demonstrated by realizing eight parallel traps at a total removal rate of 0.9 μL min(-1) (measured for nitrogen).     

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian cells s(-1) with 37% sorting purity, 90% cell viability in enrichment mode, and >90% purity in high purity mode at 1500 cells s(-1) or 3000 beads s(-1). Fast switching (30 μs) and a small perturbation volume (~90 pL) is achieved by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.     

Pipette-friendly laminar flow patterning for cell-based assays

Laminar flow patterning (LFP) is a characteristic method of microfluidic systems that allows two (or more) different solutions to flow side-by-side in a channel without convective mixing. This fluid behavior can be used to pattern cell suspensions, particles, and treatments as well as to create chemical gradients. LFP is typically implemented using syringe pumps and, for this reason, is most effective in constant flow scenarios such as long-term gradient generation. However, the complexity of using syringe pumps for patterning cell suspensions typically makes it a less attractive option than other standard patterning methods. We present a passive microfluidic method that enables short-term LFP of multiple fluids using a single pipette and allows each sample to be loaded in any sequence, at any point in time relative to one another. The proposed method is well-suited for cell-based assays, reduces the complexity of LFP to be on a similar level as other cell patterning methods, can be scaled to include more than two streams of fluid, and enables arrays of individually addressable devices for LFP on a single chip.     

A membrane-based, high-efficiency, microfluidic debubbler

In many lab-on-chip applications, it is necessary to remove bubbles from the flow stream. Existing bubble removal strategies have various drawbacks such as low degassing efficiency, long degassing time, large dead volumes, sensitivity to surfactants, and the need for an external vacuum or pressure source. We report on a novel, simple, robust, passive, nozzle-type, membrane-based debubbler that can be readily incorporated into microfluidic devices for rapid degassing. The debubbler is particularly suitable to operate with microfluidic systems made with plastic. The debubbler consists of a hydrophobic, porous membrane that resembles a normally closed valve, which is forced open by the working fluid's pressure. To illustrate the operation of the debubbler, we describe its use in the context of a chip containing a bead array for immunoassays. Our debubbler was able to completely filter gas bubbles out of a segmented flow at rates up to 60 μl s(-1) mm(-2) of membrane area.     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

An effervescent reaction micropump for portable microfluidic systems

A water-activated, effervescent reaction was used to transport fluid in a controllable manner on a portable microfluidic device. The reaction between sodium bicarbonate and an organic acid, tartaric acid and/or benzoic acid, was modeled to analyze methods of controlling the generation of carbon-dioxide gas for the purposes of pumping fluids. Integration and testing of the effervescent reaction pump in a microfluidic device was made possible by using elastomeric polymers as both photopolymerizable septa and removable lids. These materials combined to enable facile access to otherwise gas-tight devices. Based on theoretical predictions for 0.33 mg of sodium bicarbonate and a stoichiometric amount of organic acid, the pumping flow rate could be varied from 0.01 microL s(-1) to 70 microL s(-1). The flow rate is controlled by adjusting any or all of the particle size of the least soluble reactant, the amount of reactants used, and the type of organic acid selected. The tartaric acid systems rapidly produce carbon dioxide; however, the gas generation rates dramatically decrease over the course of the reaction. In contrast, carbon dioxide production rate in the benzoic acid systems is lower and nearly constant for several minutes. Water activation and direct placement on a microfluidic device are key features of this micropump, which is therefore useful for portable microfluidic applications.     

Photo-actuation of liquids for light-driven microfluidics: state of the art and perspectives

Using light to control liquid motion is a new paradigm for the actuation of microfluidic systems. We review here the different principles and strategies to induce or control liquid motion using light, which includes the use of radiation pressure, optical tweezers, light-induced wettability gradients, the thermocapillary effect, photosensitive surfactants, the chromocapillary effect, optoelectrowetting, photocontrolled electroosmotic flows and optical dielectrophoresis. We analyze the performance of these approaches to control using light many kinds of microfluidic operations involving discrete pL- to μL-sized droplets (generation, driving, mixing, reaction, sorting) or fluid flows in microchannels (valve operation, injection, pumping, flow rate control). We show that a complete toolbox is now available to control microfluidic systems by light. We finally discuss the perspectives of digital optofluidics as well as microfluidics based on all optical fluidic chips and optically reconfigurable devices.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Thermal analysis of nanofluids in microfluidics using an infrared camera

We present the thermal analysis of liquid containing Al(2)O(3) nanoparticles in a microfluidic platform using an infrared camera. The small dimensions of the microchannel along with the low flow rates (less than 120 μl min(-1)) provide very low Reynolds numbers of less than 17.5, reflecting practical parameters for a microfluidic cooling platform. The heat analysis of nanofluids has never been investigated in such a regime, due to the deficiencies of conventional thermal measurement systems. The infrared camera allows non-contact, three dimensional and high resolution capability for temperature profiling. The system was studied at different w/w concentrations of thermally conductive Al(2)O(3) nanoparticles and the experiments were in excellent agreement with the computational fluid dynamics (CFD) simulations.     

Simple and cheap microfluidic devices for the preparation of monodisperse emulsions

Droplet microfluidics, which can generate monodisperse droplets or bubbles in unlimited numbers, at high speed and with complex structures, have been extensively investigated in chemical and biological fields. However, most current methods for fabricating microfluidic devices, such as glass etching, soft lithography in polydimethylsiloxane (PDMS) or assembly of glass capillaries, are usually either expensive or complicated. Here we report the fabrication of simple and cheap microfluidic devices based on patterned coverslips and microscope glass slides. The advantages of our approach for fabricating microfluidic devices lie in a simple process, inexpensive processing equipment and economical laboratory supplies. The fabricated microfluidic devices feature a flexible design of microchannels, easy spatial patterning of surface wettability, and good chemical compatibility and optical properties. We demonstrate their utilities for generation of monodisperse single and double emulsions with highly controllable flexibility.     

Microbubble generation in a co-flow device operated in a new regime

A new regime of operation of PDMS-based flow-focusing microfluidic devices is presented. We show that monodisperse microbubbles with diameters below one-tenth of the channel width (here w = 50 μm) can be produced in low viscosity liquids thanks to a strong pressure gradient in the entrance region of the channel. In this new regime bubbles are generated at the tip of a long and stable gas ligament whose diameter, which can be varied by tuning appropriately the gas and liquid flow rates, is substantially smaller than the channel width. Through this procedure the volume of the bubbles formed at the tip of the gas ligament can be varied by more than two orders of magnitude. The experimental results for the bubble diameter d(b) as function of the control parameters are accounted for by a scaling theory, which predicts d(b)/w ∝ (μ(g)/μ(l))(1/12)(Q(g)/Q(l))(5/12), where μ(g) and μ(l) indicate, respectively, the gas and liquid viscosities and Q(g) and Q(l) are the gas and liquid flow rates. As a particularly important application of our results we produce monodisperse bubbles with the appropriate diameter for therapeutic applications (d(b) ? 5 μm) and a production rate exceeding 10(5) Hz.     

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).     

Integrated three-dimensional filter separates nanoscale from microscale elements in a microfluidic chip

We report on the integration of a size-based three-dimensional filter, with micrometre-sized pores, in a commercial microfluidic chip. The filter is fabricated inside an already sealed microfluidic channel using the unique capabilities of two-photon polymerization. This direct-write technique enables integration of the filter by post-processing in a chip that has been fabricated by standard technologies. The filter is located at the intersection of two channels in order to control the amount of flow passing through the filter. Tests with a suspension of 3 μm polystyrene spheres in a Rhodamine 6G solution show that 100% of the spheres are stopped, while the fluorescent molecules are transmitted through the filter. We demonstrate operation up to a period of 25 minutes without any evidence of clogging. Preliminary validation of the device for plasma separation from whole blood is shown. Moreover, the filter can be cleaned and reused by reversing the flow.     

Microfluidic device for capillary electrochromatography-mass spectrometry

A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4-8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement.     

Microfluidic pressure sensing using trapped air compression

We have developed a microfluidic method for measuring the fluid pressure head experienced at any location inside a microchannel. The principal component is a microfabricated sealed chamber with a single inlet and no exit; the entrance to the single inlet is positioned at the location where pressure is to be measured. The pressure measurement is then based on monitoring the movement of a liquid-air interface as it compresses air trapped inside the microfabricated sealed chamber and calculating the pressure using the ideal gas law. The method has been used to measure the pressure of the air stream and continuous liquid flow inside microfluidic channels (d approximately 50 microm). Further, a pressure drop has also been measured using multiple microfabricated sealed chambers. For air pressure, a resolution of 700 Pa within a full-scale range of 700-100 kPa was obtained. For liquids, pressure drops as low as 70 Pa were obtained in an operating range from 70 Pa to 10 kPa. Since the method primarily uses a microfluidic sealed chamber, it does not require additional fabrication steps and may easily be incorporated in several lab-on-a-chip fluidic applications for laminar as well as turbulent flow conditions.