Hybrid Microfluidic Device Research Articles

Commentary on Some Recent Theses Relevant to Combating Aging: December 2019.

Theses reviewed in this issue include "Atrophied Thymus, a Tumor Reservoir for Harboring Melanoma Cells," "Evolutionary Adaptations in Developmental Signaling Pathways Underlie Regenerative Scar-Free Wound Repair in African Spiny Mouse (Genus Acomys)," "Integrated Immunoassays on Paper/Polymer Hybrid Microfluidic Devices for Low-Cost Detection of Disease Biomarkers," "RNA Regulation in the Nervous System: CircRNA Expression Changes During Aging, and Function of the Calm1 Extended 3' UTR Isoform," "The Role of Amylin in Alzheimer's Disease," and "Therapeutic Monoclonal Antibodies to Detect and Halt ATTR Cardiac Amyloidosis and Neuropathy."

Rapid and Highly Controlled Generation of Monodisperse Multiple Emulsions via a One-Step Hybrid Microfluidic Device

Multiple Emulsions (MEs) contain a drop laden with many micro-droplets. A single-step microfluidic-based synthesis process of MEs is presented to provide a rapid and controlled generation of monodisperse MEs. The design relies on the interaction of three immiscible fluids with each other in subsequent droplet formation steps to generate monodisperse ME constructs. The design is within a microchannel consists of two compartments of cross-junction and T-junction. The high shear stress at the cross-junction creates a stagnation point that splits the first immiscible phase to four jet streams each of which are sprayed to micrometer droplets surrounded by the second phase. The resulted structure is then supported by the third phase at the T-junction to generate and transport MEs. The ME formation within microfluidics is numerically simulated and the effects of several key parameters on properties of MEs are investigated. The dimensionless modeling of ME formation enables to change only one parameter at the time and analyze the sensitivity of the system to each parameter. The results demonstrate the capability of highly controlled and high-throughput MEs formation in a one-step synthesis process. The consecutive MEs are monodisperse in size which open avenues for the generation of controlled MEs for different applications.

Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells

We introduce a new method to construct microfluidic devices especially useful for bulk acoustic wave (BAW)-based manipulation of cells and microparticles. To obtain efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium in which the cells/microparticles are suspended and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW-based acoustofluidic channel fabrication. Silicon- and glass-based fabrication is typically performed in clean room facilities, generates hazardous waste, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of poly(dimethylsiloxane) (PDMS) onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low cost and effective in BAW applications. To this end, we micromachined aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and the generation of multiple focused streams in flow-through systems. Graphical abstract The aluminum acoustofluidic device and the generation of multinode focusing of particles.

Enhanced Microchip Electrophoresis Separations Combined with Electrochemical Detection Utilizing a Capillary Embedded in Polystyrene.

The ability to use microchip-based electrophoresis for fast, high-throughput separations provides researchers with a tool for close-to real time analysis of biological systems. While PDMS-based electrophoresis devices are popular, the separation efficiency is often an issue due to the hydrophobic nature of PDMS. In this study, a hybrid microfluidic capillary device was fabricated to utilize the positive features of PDMS along with the electrophoretic performance of fused silica. A capillary loop was embedded in a polystyrene base that can be coupled with PDMS microchannels at minimal dead volume interconnects. A method for cleaning out the capillaries after a wet-polishing step was devised through the use of 3D printed syringe attachment. By comparing the separation efficiency of fluorescein and CBI-glycine with both a PDMS-based serpentine device and the embedded capillary loop device, it was shown that the embedded capillary loop device maintained higher theoretical plates for both analytes. A Pd decoupler with a carbon or Pt detection electrode were embedded along with the loop allowing integration of the electrophoretic separation with electrochemical detection. A series of catecholamines were separated to show the ability to resolve similar analytes and detect redox active species. The release of dopamine and norepinephrine from PC 12 cells was also analyzed showing the compatibility of these improved microchip separations with high ionic cell buffers associated with cell culture.

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

Paper-Plastic Hybrid Microfluidic Device for Smartphone-Based Colorimetric Analysis of Urine.

In this work, a disposable paper-plastic hybrid microfluidic lab-on-a-chip (LOC) has been developed and successfully applied for the colorimetric measurement of urine by the smartphone-based optical platform using a "UrineAnalysis" Android app. The developed device was cost-effectively implemented as a stand-alone hybrid LOC by incorporating the paper-based conventional reagent test strip inside the plastic-based LOC microchannel. The LOC device quantitatively investigated the small volume (40 μL) of urine analytes for the colorimetric reaction of glucose, protein, pH, and red blood cell (RBC) in integration with the finger-actuating micropump. On the basis of our experiments, the conventional urine strip showed large deviation as the reaction time goes by, because dipping the strip sensor in a bottle of urine could not control the reaction volume. By integrating the strip sensor in the LOC device for urine analysis, our device significantly improves the time-dependent inconstancy of the conventional dipstick-based urine strip, and the smartphone app used for image analysis enhances the visual assessment of the test strip, which is a major user concern for the colorimetric analysis in point-of-care (POC) applications. As a result, the user-friendly LOC, which is successfully implemented in a disposable format with the smartphone-based optical platform, may be applicable as an effective tool for rapid and qualitative POC urinalysis.

Polymer-based microfluidic chips for isothermal amplification of nucleic acids

Creation of low-cost compact devices based on microfluidic platforms for biological and medical research depends on the degree of development and enhancement of prototyping technologies. Two designs of polymer and hybrid microfluidic devices fabricated by soft lithography and intended for isothermal amplification and polymerase chain reaction are presented in this paper. The digital helicase-dependent isothermal amplification was tested in the device containing a droplet generator. Polymerase chain reaction was carried out in the hybrid microfluidic device having ten reaction chambers. A synthesized cDNA fragment of GAPDH housekeeping gene was used as a target.

Simple reagent storage in polyester-paper hybrid microdevices for colorimetric detection

A challenge in developing microfluidic devices for fully portable and automated field analyses is the ability to integrate and store reagents in a stable form while maintaining a low cost for the system. Paper-based field-deployable devices are currently the most extensively explored platforms for field use; however, centrifugal devices offer a means for heightened device automation and portability. The basis for many field devices is chemical sensing via colorimetric reactions. Here, we describe the development of polyester-paper hybrid microfluidic devices using a fabrication method, compounded from the print, cut and laminate (PCL) technique, that allows for simple yet effective dry reagent storage for enhanced use with centrifugal or polymeric-based devices. We utilized various proof-of-principle colorimetric assays for multiple applications, including the analysis of total protein, serum albumin, cocaine, 2,4,6-trinitrotoluene (TNT) and iron content. Localized reagent integration facilitated by paper punches allowed for both reagent stability prior to reaction for storage, color stability post-reaction for image analysis, and was compatible for multi-step analyses. Qualitative and quantitative results were achieved with the broad range of sample applications trialed here, while still maintaining the ability for downstream processing.

Developing a non-fouling hybrid microfluidic device for applications in circulating tumour cell detections

Non-specific cell adsorption is a challenge in sensitive detections using microfluidic systems, such as detecting circulating tumour cells from blood samples. In this report, we present a new strategy to study the combined effects of surface hydrophilicity/hydrophobicity, electric charges and roughness on surface non-fouling properties of a PDMS/SU-8 microfluidic system. To achieve this, microchannel surfaces were modified by poly(amidoamine) generation 4 and generation 7, dendrimers that rendered surfaces negatively and positively charged at pH 7.4, respectively. Water contact angle, atomic force microscopy (AFM) and microscopy were used to characterize and confirm surface modifications, and the non-fouling performance of the resulting surfaces was tested using both live and dead CCRM-CEM cancer cells. Our results show that for live cells, electric charges of a surface is the most important factor affecting the non-fouling features of the surface in microfluidic systems; in contrast, for dead cells, surface hydrophilicity is the most important factor affecting surface non-fouling properties. However, surface roughness does not seem to be as important for both live and dead cells under the experimental conditions used in this study. These results also highlight the importance of different considerations when designing a lab-on-a-chip microfluidic system for high sensitivity biosensing and detection applications.

A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons.

C. elegans is a well-known model organism in biology and neuroscience with a simple cellular (959 cells) and nervous (302 neurons) system and a relatively homologous (40%) genome to humans. Lateral and longitudinal manipulation of C. elegans to a favorable orientation is important in many applications such as neural and cellular imaging, laser ablation, microinjection, and electrophysiology. In this paper, we describe a micro-electro-fluidic device for on-demand manipulation of C. elegans and demonstrate its application in imaging of organs and neurons that cannot be visualized efficiently under natural orientation. To achieve this, we have used the electrotaxis technique to longitudinally orient the worm in a microchannel and then insert it into an orientation and imaging channel in which we integrated a rotatable glass capillary for orientation of the worm in any desired direction. The success rates of longitudinal and lateral orientations were 76% and 100%, respectively. We have demonstrated the application of our device in optical and fluorescent imaging of vulva, uterine-vulval cell (uv1), vulB1\2 (adult vulval toroid cells), and ventral nerve cord of wild-type and mutant worms. In comparison to existing methods, the developed technique is capable of orienting the worm at any desired angle and maintaining the orientation while providing access to the worm for potential post-manipulation assays. This versatile tool can be potentially used in various applications such as neurobehavioral imaging, neuronal ablation, microinjection, and electrophysiology.

Separation of magnetic beads in a hybrid continuous flow microfluidic device

Magnetic separation of biological entities in microfluidic environment is a key task for a large number of bio-analytical protocols. In magnetophoretic separation, biochemically functionalized magnetic beads are allowed to bind selectively to target analytes, which are then separated from the background stream using a suitably imposed magnetic field. Here we present a numerical study, characterizing the performance of a magnetophoretic hybrid microfluidic device having two inlets and three outlets for immunomagnetic isolation of three different species from a continuous flow. The hybrid device works on the principle of split-flow thin (SPLITT) fractionation and field flow fractionation (FFF) mechanisms. Transport of the magnetic particles in the microchannel has been predicted following an Eulerian-Lagrangian model and using an in-house numerical code. Influence of the salient geometrical parameters on the performance of the separator is studied by characterizing the particle trajectories and their capture and separation indices. Finally, optimum channel geometry is identified that yields the maximum capture efficiency and separation index.

Hybrid microfluidics combined with active and passive approaches for continuous cell separation.

Microfluidics, which is classified as either active or passive, is capable of separating cells of interest from a complex and heterogeneous sample. Active methods utilise external fields such as electric, magnetic, acoustic, and optical to drive cells for separation, while passive methods utilise channel structures, intrinsic hydrodynamic forces, and steric hindrances to manipulate cells. However, when processing complex biological samples such as whole blood with rare cells, separation with a single module microfluidic device is difficult. Hybrid microfluidics is an emerging technique, which utilises active and passive methods whilst fulfilling higher requirements for stable performance, versatility, and convenience, including (i) the ability to process multi-target cells, (ii) enhanced ability for multiplexed separation, (iii) higher sensitivity, and (iv) tunability for a wider operational range. This review introduces the fundamental physics and typical formats for subclasses of hybrid microfluidic devices based on their different physical fields; presents current examples of cell sorting to highlight the advantage and usefulness of hybrid microfluidics on biomedicine, and then discusses the challenges and perspective of future development and the promising direction of research in this field.

Surface micromachining of polydimethylsiloxane for microfluidics applications.

Polydimethylsiloxane (PDMS) elastomer has emerged as one of the most frequently applied materials in microfluidics. However, precise and large-scale surface micromachining of PDMS remains challenging, limiting applications of PDMS for microfluidic structures with high-resolution features. Herein, surface patterning of PDMS was achieved using a simple yet effective method combining direct photolithography followed by reactive-ion etching (RIE). This method incorporated a unique step of using oxygen plasma to activate PDMS surfaces to a hydrophilic state, thereby enabling improved adhesion of photoresist on top of PDMS surfaces for subsequent photolithography. RIE was applied to transfer patterns from photoresist to underlying PDMS thin films. Systematic experiments were conducted in the present work to characterize PDMS etch rate and etch selectivity of PDMS to photoresist as a function of various RIE parameters, including pressure, RF power, and gas flow rate and composition. We further compared two common RIE systems with and without bias power and employed inductively coupled plasma and capacitively coupled plasma sources, respectively, in terms of their PDMS etching performances. The RIE-based PDMS surface micromachining technique is compatible with conventional Si-based surface and bulk micromachining techniques, thus opening promising opportunities for generating hybrid microfluidic devices with novel functionalities.

Light-harvesting Ni/TiO2 nanotubes as photo-electrocatalyst for alcohol oxidation in alkaline media

Anodic photo-electrocatalysts comprising anodically grown TiO2 nanotubes (TiO2-NT) with an average inner diameter of 70nm and lengths of 7, 15 and 30μm with nanoparticulate electrodeposited Ni species (namely NiO, Ni(OH)2) were synthesized and evaluated for its use as anode for direct methanol fuel cell. The Ni deposit with an average diameter of 200nm was obtained by cyclic voltammetry from a modified acid Watts bath. Morphology, composition, crystallinity and optical properties were investigated by SEM, EDS, XRD, Raman and diffuse reflectance spectroscopy. UV–vis irradiation coming from a Xe lamp on the anodes increased the current density by 24% in half-cell configuration. Finally, a microfluidic fuel cell was assembled and tested using methanol as fuel reached an OCP of 0.7V. This finding suggests that such composite materials are promising alternatives for the development of a new generation of hybrid-microfluidic devices.

Versatile microfluidic complement fixation test for disease biomarker detection

The complement fixation test (CFT) is a serological test that can be used to detect the presence of specific antibodies or antigens to diagnose infections, particularly diseases caused by microbes that are not easily detected by standard culture methods. We report here, for the first time, a poly(dimethylsiloxane) (PDMS)/glass slide hybrid microfluidic device that was used to manipulate the solution compartment and communication within the microchannel to establish sampler and indicator systems of CFT. Two types of on-chip CFT, solution-based and solid phase agar-based assays, were successfully demonstrated for biomarker carcinoembryonic antigen (CEA) and recombinant avian influenza A (rH7N9) virus protein detection. In addition, the feasibility of the on-chip CFT in assaying real biopsy was successfully demonstrated by specifically detecting rH7N9 and CEA in human serum. The results demonstrated that the miniaturized assay format significantly reduced the assay time and sample consumption. Exemption from protein immobilization, blocking, complicated washing steps and expensive enzyme/fluorescein conjugates highlights the merits of on-chip CFT over ELISA. Most attractively, the on-chip agar-based CFT results can be imaged and analysed by smartphone, strengthening its point-of-care application potential. We anticipate that the on-chip CFT reported herein will be a useful supplemental or back-up tool for on-chip immunoassays such as ELISA for disease diagnosis and food inspection.

An integrated hybrid microfluidic device for oviposition-based chemical screening of adult Drosophila melanogaster.

Chemical screening using Drosophila melanogaster (the fruit fly) is vital in drug discovery, agricultural, and toxicological applications. Oviposition (egg laying) on chemically-doped agar plates is an important read-out metric used to quantitatively assess the biological fitness and behavioral responses of Drosophila. Current oviposition-based chemical screening studies are inaccurate, labor-intensive, time-consuming, and inflexible due to the manual chemical doping of agar. In this paper, we have developed a novel hybrid agar-polydimethylsiloxane (PDMS) microfluidic device for single- and multi-concentration chemical dosing and on-chip oviposition screening of free-flying adult stage Drosophila. To achieve this, we have devised a novel technique to integrate agar with PDMS channels using ice as a sacrificial layer. Subsequently, we have conducted single-chemical toxicity and multiple choice chemical preference assays on adult Drosophila melanogaster using zinc and acetic acid at various concentrations. Our device has enabled us to 1) demonstrate that Drosophila is capable of sensing the concentration of different chemicals on a PDMS-agar microfluidic device, which plays significant roles in determining oviposition site selection and 2) investigate whether oviposition preference differs between single- and multi-concentration chemical environments. This device may be used to study fundamental and applied biological questions in Drosophila and other egg laying insects. It can also be extended in design to develop sophisticated and dynamic chemical dosing and high-throughput screening platforms in the future that are not easily achievable with the existing oviposition screening techniques.

Developing an ultra non-fouling SU-8 and PDMS hybrid microfluidic device by poly(amidoamine) engraftment

A new strategy to prepare an ultra non-fouling SU-8/poly(dimethylsiloxane) (PDMS) hybrid microfluidic system is presented. We report a simple method to bond both SU-8 and PDMS surfaces to prepare the hybrid microfluidic device and then simultaneously modify both SU-8 and PDMS surfaces using poly(amidoamine) (PAMAM), a highly hydrophilic dendrimer to enhance non-fouling properties of the hybrid microfluidic channel. Water contact angle, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) are used to characterize and confirm surface modifications. Finally the non-fouling performance of the resulting microfluidic system is tested using both microbeads and Escherichia coli O157:H7 bacterial cells. We demonstrate that the obtained hybrid microfluidic system shows a significant microbead adsorption suppression of 99.7%, an ultra non-fouling performance that has not been reported in the literature before; in addition, 95% bacteria surface adsorption suppression is also obtained. Considering the significantly improved non-fouling performance and the ease of preparing the hybrid microfluidic system, we anticipate that this strategy will find wide applications in biosensing and detection.

A room-temperature bonding technique for the packaging of hydrogel-based hybrid microfluidic devices

We present a room-temperature bonding technique which enables the sealing of micromolded water-swollen poly-2-hydroxy ethyl methacrylate (PHEMA)-based hydrogel modules to thermoplastic and elastomeric platforms. The core mechanism of the proposed method is to favor the formation of hydrogen bonds at the interface between the surface-reactive hydrated hydrogel components and the functionalized hydrophobic materials, by contacting the polymeric layers via a mild compression. Plasma oxygen and silanization processes using 3-amino-propyl trimethoxy silane have been optimized to generate surface hydroxyl functionalities on poly-methyl methacrylate (PMMA) and poly-dimethyl siloxane (PDMS) platforms which can react with the OH groups’ enriched surface of the microstructured PHEMA. The resulting microsystems can operate in a continuous perfusion mode while being soaked in water to keep the hydrogel hydrated. The method could also be applied to bond PHEMA thin layers (300 μm thick) to PMMA and PDMS microfluidic components. The sealing properties of the produced devices were demonstrated by liquid leakage tests and further validated by hydration–dehydration cycles of the systems and by monitoring methylene blue diffusion through the hydrogel matrix at the modules interface. The presented technique is suitable for cells-based hybrid materials microfluidic devices rapid prototyping.

A versatile PDMS/paper hybrid microfluidic platform for sensitive infectious disease diagnosis.

Bacterial meningitis is a serious health concern worldwide. Given that meningitis can be fatal and many meningitis cases occurred in high-poverty areas, a simple, low-cost, highly sensitive method is in great need for immediate and early diagnosis of meningitis. Herein, we report a versatile and cost-effective polydimethylsiloxane (PDMS)/paper hybrid microfluidic device integrated with loop-mediated isothermal amplification (LAMP) for the rapid, sensitive, and instrument-free detection of the main meningitis-causing bacteria, Neisseria meningitidis (N. meningitidis). The introduction of paper into the microfluidic device for LAMP reactions enables stable test results over a much longer period of time than a paper-free microfluidic system. This hybrid system also offers versatile functions, by providing not only on-site qualitative diagnostic analysis (i.e., a yes or no answer), but also confirmatory testing and quantitative analysis in laboratory settings. The limit of detection of N. meningitidis is about 3 copies per LAMP zone within 45 min, close to single-bacterium detection sensitivity. In addition, we have achieved simple pathogenic microorganism detection without a laborious sample preparation process and without the use of centrifuges. This low-cost hybrid microfluidic system provides a simple and highly sensitive approach for fast instrument-free diagnosis of N. meningitidis in resource-limited settings. This versatile PDMS/paper microfluidic platform has great potential for the point of care (POC) diagnosis of a wide range of infectious diseases, especially for developing nations.

Microdevice-based cell therapy for age-related macular degeneration.

This chapter reports the application of a micromachined parylene-C device as an artificial Bruch's membrane for the stem cell-based therapy of age-related macular degeneration. The feasibility of parylene-C as a substitute substrate material is demonstrated by evaluating the permeability of membranes of submicron thicknesses. It has been found that parylene-C membranes thinner than 0.3 µm possess similar molecular weight exclusion limit and nutrient diffusion flux to that of the healthy human Bruch's membrane. This conclusion is further validated by the in vitro perfusion cell viability test. Since the submicron parylene-C itself is difficult to handle, we design a mesh-supported submicron parylene membrane (MSPM) to provide sufficient mechanical support. This MSPM can support the growth of retinal pigment epithelial (RPE) cells in a monolayer with well-polarized morphology. Human embryonic stem cell-derived H9-RPE cells are cultured in vitro on the MSPM for one month before the implantation of the MSPM into the rat's retina. To facilitate the surgical implantation, a parylene-C/SU-8 hybrid microfluidic device is designed as an inserter. Histological studies with hematoxylin-eosin staining and immunofluorescence staining show that the implanted RPE cells adhere well to the artificial Bruch's membrane and are able to maintain high viability and normal morphology in vivo.