Systematic characterization of degas-driven flow for poly(dimethylsiloxane) microfluidic devices

Degas-driven flow is a novel phenomenon used to propel fluids in poly(dimethylsiloxane) (PDMS)-based microfluidic devices without requiring any external power. This method takes advantage of the inherently high porosity and air solubility of PDMS by removing air molecules from the bulk PDMS before initiating the flow. The dynamics of degas-driven flow are dependent on the channel and device geometries and are highly sensitive to temporal parameters. These dependencies have not been fully characterized, hindering broad use of degas-driven flow as a microfluidic pumping mechanism. Here, we characterize, for the first time, the effect of various parameters on the dynamics of degas-driven flow, including channel geometry, PDMS thickness, PDMS exposure area, vacuum degassing time, and idle time at atmospheric pressure before loading. We investigate the effect of these parameters on flow velocity as well as channel fill time for the degas-driven flow process. Using our devices, we achieved reproducible flow with a standard deviation of less than 8% for flow velocity, as well as maximum flow rates of up to 3 nL∕s and mean flow rates of approximately 1-1.5 nL∕s. Parameters such as channel surface area and PDMS chip exposure area were found to have negligible impact on degas-driven flow dynamics, whereas channel cross-sectional area, degas time, PDMS thickness, and idle time were found to have a larger impact. In addition, we develop a physical model that can predict mean flow velocities within 6% of experimental values and can be used as a tool for future design of PDMS-based microfluidic devices that utilize degas-driven flow.     

Polydimethyl siloxane wet etching for three dimensional fabrication of microneedle array and high-aspect-ratio micropillars

Among various transdermal drug delivery (TDD) approaches, utilizing the microneedles (MNs) not only can penetrate the skin but also deliver the drug with reduced tissue damage, reduced pain, and no bleeding. However, the MNs with larger height are required to overcome the skin barrier for effective TDD. Unlike 2D patterning, etching polydimethyl siloxane (PDMS) micropillars for fabrication of 3D microstructures is presented. The PDMS micropillars were first constructed by casting PDMS on the computer numerical control-machined cylindrical microwells, which then went through etching process to obtain the MNs for subsequent fabrication of polymer MNs or high aspect ratio micropillars.     

A droplet acoustofluidic platform for time-controlled microbead-based reactions

Droplet microfluidics is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.     

Simple surface modification of poly(dimethylsiloxane) for DNA hybridization

Here, we present a simple chemical modification of poly(dimethylsiloxane) (PDMS) by curing a mixture of 2 wt% undecylenic acid (UDA) in PDMS prepolymer on a gold-coated glass slide. This gold slide had been previously pretreated with a self-assembled hydrophilic monolayer of 3-mercaptopropionic acid (MPA). During curing of the UDA∕PDMS prepolymer, the hydrophilic UDA carboxyl moieties diffuses toward the hydrophilic MPA carboxyl moieties on the gold surface. This diffusion of the UDA within the PDMS prepolymer to the surface is a direct result of surface energy minimization. Once completely cured, the PDMS is peeled off the gold substrate, thereby exposing the interfacial carboxyl groups. These groups are then available for subsequent attachment of 5(')-amino terminated DNA oligonucleotides via amide linkages. Our results show that the covalently tethered oligonucleotides can successfully capture fluorescein-labeled complementary oligonucleotides via hybridization, which are visualized using fluorescence microscopy.     

Patterning nanowire and micro-∕nanoparticle array on micropillar-structured surface: Experiment and modeling

DNA molecules in a solution can be immobilized and stretched into a highly ordered array on a solid surface containing micropillars by molecular combing technique. However, the mechanism of this process is not well understood. In this study, we demonstrated the generation of DNA nanostrand array with linear, zigzag, and fork-zigzag patterns and the microfluidic processes are modeled based on a deforming body-fitted grid approach. The simulation results provide insights for explaining the stretching, immobilizing, and patterning of DNA molecules observed in the experiments.     

Applications of electrowetting-on-dielectric (EWOD) technology for droplet digital PCR

Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade.     

High throughput fabrication of disposable nanofluidic lab-on-chip devices for single molecule studies

An easy method is introduced allowing fast polydimethylsiloxane (PDMS) replication of nanofluidic lab-on-chip devices using accurately fabricated molds featuring cross-sections down to 60 nm. A high quality master is obtained through proton beam writing and UV lithography. This master can be used more than 200 times to replicate nanofluidic devices capable of handling single DNA molecules. This method allows to fabricate nanofluidic devices through simple PDMS casting. The extensions of YOYO-1 stained bacteriophage T4 and λ−DNA inside these nanochannels have been investigated using fluorescence microscopy and follow the scaling prediction of a large, locally coiled polymer chain confined in nanochannels.     

An equipment-free polydimethylsiloxane microfluidic spotter for fabrication of microarrays

This paper presents a low-cost, power-free, and easy-to-use spotter system for fabrication of microarrays. The spotter system uses embedded dispensing microchannels combined with a polydimethylsiloxane (PDMS) membrane containing regular arrays of well-defined thru-holes to produce precise, uniform DNA or protein microarrays for disease diagnosis or drug screening. Powered by pre-evacuation of its PDMS substrate, the spotter system does not require any additional components or external equipment for its operation, which can potentially allow low-cost, high-quality microarray fabrication by minimally trained individuals. Polyvinylpyrrolidone was used to modify the PDMS surface to prevent protein adsorption by the microchannels. Experimental results indicate that the PDMS spotter shows excellent printing performance for immobilizing proteins. The measured coefficient of variation (CV) of the diameter of 48 spots was 2.63% and that of the intensity within one array was 2.87%. Concentration gradient experiments revealed the superiority of the immobilization density of the PDMS spotter over the conventional pin-printing method. Overall, this low-cost, power-free, and easy-to-use spotting system provides an attractive new method to fabricate microarrays.     

Study on surface properties of PDMS microfluidic chips treated with albumin

Electrokinetic properties and morphology of PDMS microfluidic chips intended for bioassays are studied. The chips are fabricated by a casting method followed by polymerization bonding. Microchannels are coated with 1% solution of bovine serum albumin (BSA) in Tris buffer. Albumin passively adsorbs on the PDMS surface. Electrokinetic characteristics (electro-osmotic velocity, electro-osmotic mobility, and zeta potential) of the coated PDMS channels are experimentally determined as functions of the electric field strength and the characteristic electrolyte concentration. Atomic force microscopy (AFM) analysis of the surface reveals a “peak and ridge” structure of the protein layer and an imperfect substrate coating. On the basis of the AFM observation, several topologies of the BSA-PDMS surface are proposed. A nonslip mathematical model of the electro-osmotic flow is then numerically analyzed. It is found that the electrokinetic characteristics computed for a channel with the homogeneous distribution of a fixed electric charge do not fit the experimental data. Heterogeneous distribution of the fixed electric charge and the surface roughness is thus taken into account. When a flat PDMS surface with electric charge heterogeneities is considered, the numerical results are in very good agreement with our experimental data. An optimization analysis finally allowed the determination of the surface concentration of the electric charge and the degree of the PDMS surface coating. The obtained findings can be important for correct prediction and possibly for robust control of behavior of electrically driven PDMS microfluidic chips. The proposed method of the electro-osmotic flow analysis at surfaces with a heterogeneous distribution of the surface electric charge can also be exploited in the interpretation of experimental studies dealing with protein-solid phase interactions or substrate coatings.     

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila''s oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surface-modified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations.     

A numerical study on the dynamics of droplet formation in a microfluidic double T-junction

In this study, droplet formations in microfluidic double T-junctions (MFDTD) are investigated based on a two-dimensional numerical model with volume of fluid method. Parametric ranges for generating alternating droplet formation (ADF) are identified. A physical background responsible for the ADF is suggested by analyzing the dynamical stability of flow system. Since the phase discrepancy between dispersed flows is mainly caused by non-symmetrical breaking of merging droplet, merging regime becomes the alternating regime at appropriate conditions. In addition, the effects of channel geometries on droplet formation are studied in terms of relative channel width. The predicted results show that the ADF region is shifted toward lower capillary numbers when channel width ratio is less than unity. The alternating droplet size increases with the increase of channel width ratio. When this ratio reaches unity, alternating droplets can be formed at very high water fraction (wf = 0.8). The droplet formation in MFDTD depends significantly on the viscosity ratio, and the droplet size in ADF decreases with the increase of the viscosity ratio. The understanding of underlying physics of the ADF phenomenon is useful for many applications, including nanoparticle synthesis with different concentrations, hydrogel bead generation, and cell transplantation in biomedical therapy.     

Focusing and trapping of DNA molecules by head-on ac electrokinetic streaming through join asymmetric polarization

In this work, invoking join asymmetric ac polarization using double half-quadrupole electrodes in a symmetric arrangement, we demonstrate a head-on ac electro-osmotic streaming capable of focusing and trapping DNA molecules efficiently. This is manifested by the observation that picomolar DNA molecules can be trapped into a large crosslike spot with at least an order of magnitude concentration enhancement within just half a minute. We identify that the phenomenon is a combined result of the formation of two prefocused DNA jets flowing toward each other, dipole-induced attraction between focused DNA molecules, and dielectrophoretic trap on the spot. With an additional horizontal pumping, we observe that the trap can transform into a peculiar pitchfork streaming capable of continuous collection and long-distance transport of concentrated DNA molecules. We also show that the same electrode design can be used to direct assembly of submicrometer particles. This newly designed microfluidic platform not only has potentials in enhancing detection sensitivity and facilitating functional assembly for on-chip analysis but also provides an added advantage of transporting target molecules in a focused and continuous manner.     

Simulation of single DNA molecule stretching and immobilization in a de-wetting two-phase flow over micropillar-patterned surface

We investigate single DNA stretching dynamics in a de-wetting flow over micropillars using Brownian dynamics simulation. The Brownian dynamics simulation is coupled with transient flow field computation through a numerical particle tracking algorithm. The droplet formation on the top of the micropillar during the de-wetting process creates a flow pattern that allows DNA to stretch across the micropillars. It is found that DNA nanowire forms if DNA molecules could extend across the stagnation point inside the connecting water filament before its breakup. It also shows that DNA locates closer to the top wall of the micropillar has higher chance to enter the flow pattern of droplet formation and thus has higher chance to be stretched across the micropillars. Our simulation tool has the potential to become a design tool for DNA manipulation in complex biomicrofluidic devices.     

Molecular diffusion analysis of dynamic blood flow and plasma separation driven by self-powered microfluidic devices

Integration of microfluidic devices with pressure-driven, self-powered fluid flow propulsion methods has provided a very effective solution for on-chip, droplet blood testing applications. However, precise understanding of the physical process governing fluid dynamics in polydimethylsiloxane (PDMS)-based microfluidic devices remains unclear. Here, we propose a pressure-driven diffusion model using Fick''s law and the ideal gas law, the results of which agree well with the experimental fluid dynamics observed in our vacuum pocket-assisted, self-powered microfluidic devices. Notably, this model enables us to precisely tune the flow rate by adjusting two geometrical parameters of the vacuum pocket. By linking the self-powered fluid flow propulsion method to the sedimentation, we also show that direct plasma separation from a drop of whole blood can be achieved using only a simple construction without the need for external power sources, connectors, or a complex operational procedure. Finally, the potential of the vacuum pocket, along with a removable vacuum battery to be integrated with non-PDMS microfluidic devices to drive and control the fluid flow, is demonstrated.     

Polyelectrolyte multilayer surface functionalization of poly(dimethylsiloxane) (PDMS) for reduction of yeast cell adhesion in microfluidic devices

Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.     

A novel actuation method of transporting droplets by using electrical charging of droplet in a dielectric fluid

We evaluate the feasibility of manipulating droplets in two dimensions by exploiting Coulombic forces acting on conductive droplets immersed in a dielectric fluid. When a droplet suspended in an immiscible fluid is located near an electrode under a dc voltage, the droplet can be charged by direct contact, by charge transfer along an electrically conducting path, or by both mechanisms. This process is called electrical charging of droplet (ECOD). This charged droplet may then be transported rapidly by exploiting Coulombic forces. We experimentally demonstrate electrical actuation of a charged droplet by applying voltage sequences. A charged droplet is two dimensionally actuated by following the direction of the electrical field signal. The droplet does not contact the surface of the microfluidic chip when it moves. This characteristic is very advantageous because treatments of the substrate surfaces of microfluidic chip become simpler. In order to test the feasibility of using ECOD in a droplet-based microreactor, electrocoalescence of two oppositely charged droplets is also studied. When two droplets approach each other due to Coulombic attraction, a liquid bridge is formed between them. We postulate that if the applied electric field is weaker than a certain critical level, the two droplets coalesce instantaneously when the charges are exchanged and redistributed through this liquid bridge.     

Study for optical manipulation of a surfactant-covered droplet using lattice Boltzmann method

In this study, we simulated deformation and surfactant distribution on the interface of a surfactant-covered droplet using optical tweezers as an external source. Two optical forces attracted a single droplet from the center to both sides. This resulted in an elliptical shape deformation. The droplet deformation was characterized as the change of the magnitudes of surface tension and optical force. In this process, a non-linear relationship among deformation, surface tension, and optical forces was observed. The change in the local surfactant concentration resulting from the application of optical forces was also analyzed and compared with the concentration of surfactants subjected to an extensional flow. Under the optical force influence, the surfactant molecules were concentrated at the droplet equator, which is totally opposite to the surfactants behavior under extensional flow, where the molecules were concentrated at the poles. Lastly, the quasi-equilibrium surfactant distribution was obtained by combining the effects of the optical forces with the extensional flow. All simulations were executed by the lattice Boltzmann method which is a powerful tool for solving micro-scale problems.     

Biochip∕laser cell deposition system to assess polarized axonal growth from single neurons and neuron∕glia pairs in microchannels with novel asymmetrical geometries

Axon path-finding plays an important role in normal and pathogenic brain development as well as in neurological regenerative medicine. In both scenarios, axonal growth is influenced by the microenvironment including the soluble molecules and contact-mediated signaling from guiding cells and cellular matrix. Microfluidic devices are a powerful tool for creating a microenvironment at the single cell level. In this paper, an asymmetrical-channel-based biochip, which can be later incorporated into microfluidic devices for neuronal network study, was developed to investigate geometric as well as supporting cell control of polarized axonal growth in forming a defined neuronal circuitry. A laser cell deposition system was used to place single cells, including neuron-glia pairs, into specific microwells of the device, enabling axonal growth without the influence of cytophilic∕phobic surface patterns. Phase microscopy showed that a novel "snag" channel structure influenced axonal growth in the intended direction 4:1 over the opposite direction. In heterotypic experiments, glial cell influence over the axonal growth path was observed with time-lapse microscopy. Thus, it is shown that single cell and heterotypic neuronal path-finding models can be developed in laser patterned biochips.     

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.     

Stretching of DNA confined in nanochannels with charged walls

There is currently a growing interest in control of stretching of DNA inside nanoconfined regions due to the possibility to analyze and manipulate single biomolecules for applications such as DNA mapping and barcoding, which are based on stretching the DNA in a linear fashion. In the present work, we couple Finite Element Methods and Monte Carlo simulations in order to study the conformation of DNA molecules confined in nanofluidic channels with neutral and charged walls. We find that the electrostatic forces become more and more important when lowering the ionic strength of the solution. The influence of the nanochannel cross section geometry is also studied by evaluating the DNA elongation in square, rectangular, and triangular channels. We demonstrate that coupling electrostatically interacting walls with a triangular geometry is an efficient way to stretch DNA molecules at the scale of hundreds of nanometers. The paper reports experimental observations of λ-DNA molecules in poly(dimethylsiloxane) nanochannels filled with solutions of different ionic strength. The results are in good agreement with the theoretical predictions, confirming the crucial role of the electrostatic repulsion of the constraining walls on the molecule stretching.