Electroosmotic flow patterning using microfluidic delay loops

A theoretical and experimental investigation of alternating electroosmotic flow patterns by means of specially designed delay loops is presented. Using elementary methods of compact network modeling and detailed FEM simulations the flow behavior and, in particular, the rearrangement of sample plugs is modeled. The proposed designs rely on flow splitting in combination with electroosmotic delay loops leading to a runtime difference or phase shift between two sub-streams. Due to this phase shift, a new fluid interface is generated at the merging point. The approach is experimentally validated by injection of a Rhodamine 6G solution into an aqueous sodium tetraborate buffer.     

Size-controlled flow synthesis of gold nanoparticles using a segmented flow microfluidic platform

Segmented flow is often used in the synthesis of nanomaterials to achieve narrow particle size distribution. The narrowness of the distribution is commonly attributed to the reduced dispersion associated with segmented flows. On the basis of the analysis of flow fields and the resulting particle size distribution, we demonstrate that it is the slip velocity between the two fluids and internal mixing in the continuous-phase slugs that govern the nature of the particle size distribution. The reduction in the axial dispersion has less impact on particle growth and hence on the particle size distribution. Synthesis of gold nanoparticles from HAuCl(4) with rapid reduction by NaBH(4) serves as a model system. Rapid reduction yields gold nuclei, which grow by agglomeration, and it is controlled by the interaction of the nuclei with local flow. Thus, the difference in the physical properties of the two phases and the inlet flow rates ultimately control the particle growth. Hence, a careful choice of continuous and dispersed phases is necessary to control the nanoparticle size and size distribution.     

Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets

Protein crystallization is a major bottleneck in determining tertiary protein structures from genomic sequence data. This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization droplet. The droplets are formed by mixing protein, precipitant, and additive stock solutions in variable ratios in a flow of water-immiscible fluids inside microchannels. Each droplet represents a discrete trial testing different conditions. The system has been validated by crystallization of several water-soluble proteins.     

Effect of surface nanotopography on immunoaffinity cell capture in microfluidic devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.     

Electrokinetic flow control in microfluidic chips using a field-effect transistor

A field-effect transistor is developed to control flow in microfluidic chips by modifying the surface charge condition. In this investigation, zeta potential at a particular location is altered locally by applying a gate voltage, while zeta potential at other locations is maintained at its original value. This non-uniform zeta potential results in a secondary electroosmotic flow in the lateral direction, which is used for flow control in microgeometries. Here, microchannel structures and field-effect transistors are formed on polydimethylsiloxane (PDMS) using soft lithography techniques, and a micro particle image velocimetry technique is used to obtain high resolution velocity distribution in the controlled region. The flow control is observed at relatively low gate voltage (less than 50 V), and this local flow control is primarily due to current leakage through the interface between PDMS and glass layers. A leakage capacitance model is introduced to estimate the modified zeta potential for the straight channel case, and excellent agreement is obtained between the predicted and experimental zeta potential results. This leakage-current based field-effect is then applied to a T-channel junction to control flow in the branch channel. Experiments show that the amount of discharge in the branch channel can be controlled by modulating gate voltage.     

Sub-second isoelectric focusing in free flow using a microfluidic device

Using a microfabricated chip with a bed volume of 0.2 microL we demonstrate the validity of the scaling laws for molecular mass transport of isoelectric focusing (IEF) in free flow. Nano- or microlitre sample volumes can be concentrated within 430 ms by a factor of up to 400. These very fast performances make the chip applicable to proteomic analysis and for continuous monitoring of biochemical processes.     

Uniform mixing in paper-based microfluidic systems using surface acoustic waves

Paper-based microfluidics has recently received considerable interest due to their ease and low cost, making them extremely attractive as point-of-care diagnostic devices. The incorporation of basic fluid actuation and manipulation schemes on paper substrates, however, afford the possibility to extend the functionality of this simple technology to a much wider range of typical lab-on-a-chip operations, given its considerable advantages in terms of cost, size and integrability over conventional microfluidic substrates. We present a convective actuation mechanism in a simple paper-based microfluidic device using surface acoustic waves to drive mixing. Employing a Y-channel structure patterned onto paper, the mixing induced by the 30 MHz acoustic waves is shown to be consistent and rapid, overcoming several limitations associated with its capillary-driven passive mixing counterpart wherein irreproducibilities and nonuniformities are often encountered in the mixing along the channel--capillary-driven passive mixing offers only poor control, is strongly dependent on the paper's texture and fibre alignment, and permits backflow, all due to the scale of the fibres being significant in comparison to the length scales of the features in a microfluidic system. Using a novel hue-based colourimetric technique, the mixing speed and efficiency is compared between the two methods, and used to assess the effects of changing the input power, channel tortuousity and fibre/flow alignment for the acoustically-driven mixing. The hue-based technique offers several advantages over grayscale pixel intensity analysis techniques in facilitating quantification without limitations on the colour contrast of the samples, and can be used, for example, for quantification in on-chip immunochromatographic assays.     

Heck reactions using segmented flow conditions

Various Heck couplings have been carried out using segmented flow conditions to accelerate the reactions. Aryl iodides and aryl bromides as well as anilines in diazonium-type Heck reactions have been used successfully.     

Mapping vortex-like hydrodynamic flow in microfluidic networks using fluorescence correlation spectroscopy

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (μTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (τ_F) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro -reactor and -mixer.     

Continuous concentration of bacteria in a microfluidic flow cell using electrokinetic techniques

A novel method for the concentration of bacterial solutions is presented that implements electrokinetic techniques, zone electrophoresis (ZE) and isoelectric focusing (IEF), in a microfluidic device. The method requires low power (< 3e-5 W) and can be performed continuously on a flowing stream. The device consists of two palladium electrodes held in a flow cell constructed from layers of polymeric film held together by a pressure-sensitive adhesive. Both ZE and IEF are performed with carrier-free solutions in devices in which the electrodes are in intimate contact with the sample fluid. IEF experiments were performed using natural pH gradients; no carrier ampholyte solution was required. Experiments performed in buffer alone resulted in significant electroosmotic flow. Pretreatment of the sample chamber with bleach followed by a concentrated solution of cationic detergent effectively suppressed electroosmotic flow.     

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.     

Determination of single cell surface protein expression using a tagless microfluidic method

We describe a method to detect the expression of a surface protein in single cells without prior labeling or manipulation using a microfluidic device. When the protein is expressed on a cell surface, it undergoes transient bond formation with an immobilized ligand as the cell is pumped through a microfluidic channel, resulting in a specific decrease in the cell's velocity. We were able to detect the expression of interleukin 13 receptor alpha 2 (IL13Rα2) differentially expressed on LM2 cells, a subline of MDA-MB-231 human breast cancer cells with a unique lung metastatic capability. The detection of cells with high expression of the protein was near 100% sensitive and 100% specific. We also provided proof of principle of multiplexing by tracking the same cell over two, differentially-coated patches. The method is non-destructive and cells can be collected for reanalysis. The system can identify positive cells in a cell mixture. This method will have a potential impact in analyzing cancer cells when only a few are available, such as the case with needle aspirates and when labeling and manipulation result in cell loss.     

Hydrophilic surface modification of cyclic olefin copolymer microfluidic chips using sequential photografting

The plastic material known as cyclic olefin copolymer (COC) is a useful substrate material for fabricating microfluidic devices due to its low cost, ease of fabrication, excellent optical properties, and resistance to many solvents. However, the hydrophobicity of native COC limits its use in bioanalytical applications. To increase surface hydrophilicity and reduce protein adsorption, COC surfaces were photografted with poly(ethylene glycol) methacrylate (PEGMA) using a two-step sequential approach: covalently-bound surface initiators were formed in the first step and graft polymerization of PEGMA was then carried out from these sites in the second step. Contact angle measurements were used to monitor and quantify the changes in surface hydrophilicity as a function of grafting conditions. As water droplet contact angles decreased from 88 degrees for native COC to 45 degrees for PEGMA-grafted surfaces, protein adsorption was also reduced by 78% for the PEGMA-modified COC microchannels as determined by a fluorescence assay. This photografting technique should enable the use of COC microdevices in a variety of bioanalytical applications that require minimal nonspecific adsorption of biomolecules.     

Integrated surface modification of fully polymeric microfluidic devices using living radical photopolymerization chemistry

Surface modification using living radical polymerization (LRP) chemistry is a powerful technique for surface modification of polymeric substrates. This research demonstrates the ability to use LRP as a polymer substrate surface‐modification platform for covalently grafting polymer chains in a spatially and temporally controlled fashion. Specifically, dithiocarbamate functionalities are introduced onto polymer surfaces using tetraethylthiuram disulfide. This technique enables integration of LRP‐based grafting for the development of an integrated, covalent surface‐modification method for microfluidic device construction. The unique photolithographic method enables construction of devices that are not substrate‐limited. To demonstrate the utility of this approach, both controlled fluid flow and cell patterning applications were demonstrated upon modification with various chemical functionalities. Specifically, poly(ethylene glycol) (375) monoacrylate and trifluoroethyl acrylate were grafted to control fluidic flow on a microfluidic device. Before patterning, surface‐functionalized samples were characterized with both goniometric and infrared spectroscopy to ensure that photografting was occurring through pendant dithiocarbamate functionalities. Near‐infrared results demonstrated conversion of grafted monomers when dithiocarbamate‐functionalized surfaces were used, as compared to dormant control surfaces. Furthermore, attenuated total reflectance/infrared spectroscopy results verified the presence of dithiocarbamate functionalities on the substrate surfaces, which were useful in grafting chains of various functionalities whose contact angles ranged from 7 to 86°. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1404–1413, 2006     

HistoFlex--a microfluidic device providing uniform flow conditions enabling highly sensitive, reproducible and quantitative in situ hybridizations

A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in combination or sequentially through the ability of the HistoFlex to monitor assays without disassembly.     

Low temperature bonding of PMMA and COC microfluidic substrates using UV/ozone surface treatment

The use of UV/ozone surface treatments for achieving low temperature bonds between PMMA and COC microfluidic substrates is evaluated. Low temperature bond strengths, approaching those of native polymer substrates bonded above their glass transition temperatures, are demonstrated for both thermoplastics. To evaluate the effects of the UV/O(3) surface treatment on the operation of bonded microfluidic devices, the relationship between UV/O(3) exposure and polymer hydrophilicity and surface chemistry are measured. Post-treatment surface chemistry is evaluated by XPS (X-ray photoelectron spectroscopy) analysis, and the stability of the treated surfaces following solvent exposure is reported. Electroosmotic flow within fabricated microchannels with modified wall surfaces is also characterized. Overall, UV/O(3) treatment is found to enable strong low temperature bonds between thermoplastic microfluidic substrates using a simple, low cost, and high throughput fabrication technology.     

Fabrication and characterization of poly(methylmethacrylate) microfluidic devices bonded using surface modifications and solvents

The fabrication of polymer microchips allows inexpensive, durable, high-throughput and disposable devices to be made. Poly(methylmethacrylate) (PMMA) microchips have been fabricated by hot embossing microstructures into the substrate followed by bonding a cover plate. Different surface modifications have been examined to enhance substrate and cover plate adhesion, including: air plasma treatment, and both acid catalyzed hydrolysis and aminolysis of the acrylate to yield carboxyl and amine-terminated PMMA surfaces. Unmodified PMMA surfaces were also studied. The substrate and cover plate adhesion strengths were found to increase with the hydrophilicity of the PMMA surface and reached a peak at 600 kN m(-2) for plasma treated PMMA. A solvent assisted system has also been designed to soften less than 50 nm of the surface of PMMA during bonding, while still maintaining microchannel integrity. The extent to which both surface modifications and solvent treatment affected the adhesion of the substrate to the cover plate was examined using nanoindentation methods. The solvent bonding system greatly increased the adhesion strengths for both unmodified and modified PMMA, with a maximum adhesion force of 5500 kN m(-2) achieved for unmodified PMMA substrates. The bond strength decreased with increasing surface hydrophilicity after solvent bonding, a trend that was opposite to what was observed for non-solvent thermal bonding.     

Continuous separation of particles using a microfluidic device equipped with flow rate control valves

We propose herein an improved microfluidic system for continuous and precise particle separation. We have previously proposed a method for particle separation called "pinched flow fractionation." Using the previously reported method, particles can be continuously separated according to differences in their diameters, simply by introducing liquid flows with and without particles into a specific microchannel structure. In this study, we incorporated PDMS membrane microvalves for flow rate control into the microfluidic device to improve the separation accuracy. By adjusting the flow rates distributed to each outlet, target particles could be precisely collected from the desired outlet. We succeeded in separating micron and submicron-size polymer particles. This method can be used widely for continuous and precise separation of various kinds of particles, and can function as an important part of microfluidic systems.     

Analysis of intercellular calcium signaling using microfluidic adjustable laminar flow for localized chemical stimulation

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300 μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26 μm with a total flow rate of 20 μL min⁻¹, which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell–cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening.     

Electroosmotic flow control in microfluidic chips using a self-assembled monolayer as the insulator of a flow field-effect transistor

A novel concept for electroosmotic flow (EOF) control in a microfluidic chip is presented by using a self-assembled monolayer as the insulator of a flow field-effect transistor. Bidirectional EOF control with mobility values of 3.4 × 10(-4) and -3.1 × 10(-4) cm(2)/V s can be attained, corresponding to the applied gate voltage at -0.8 and 0.8 V, respectively, without the addition of buffer additives. A relatively high control factor (approximately 400 × 10(-6) cm(2)/V(2) s) can be obtained. The method presented in this study offers a simple strategy to control the EOF.