Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

ZnO films for surface acoustic wave microfluidic devices

SAW devices were fabricated on c-axis oriented ZnO films grown on Si substrates. Effects of film thickness on the film microstructure and acoustic frequencies were studied. Both Rayleigh and Sezawa mode waves were detected on the devices, and their resonant frequencies were found to decrease with increase in film thickness.

ZnO films for surface acoustic wave microfluidic devices

SAW devices were fabricated on c-axis oriented ZnO films grown on Si substrates. Effects of film thickness on the film microstructure and acoustic frequencies were studied. Both Rayleigh and Sezawa mode waves were detected on the devices, and their resonant frequencies were found to decrease with increase in film thickness.

Experimental Characterization Of Electrical Current Leakage In Poly(Dimethylsiloxane) Microfluidic Devices

Poly(dimethylsiloxane) (PDMS) is usually considered as a dielectric material and the PDMS microchannel wall can be treated as an electrically insulated boundary in an applied electric field. However, in certain layouts of microfluidic networks, electrical leakage through the PDMS microfluidic channel walls may not be negligible, which must be carefully considered in the microfluidic circuit design. In this paper, we report on the experimental characterization of the electrical leakage current through PDMS microfluidic channel walls of different configurations. Our numerical and experimental studies indicate that for tens of microns thick PDMS channel walls, electrical leakage through the PDMS wall could significantly alter the electrical field in the main channel. We further show that we can use the electrical leakage through the PDMS microfluidic channel wall to control the electrolyte flow inside the microfluidic channel and manipulate the particle motion inside the microfluidic channel. More specifically, we can trap individual particles at different locations inside the microfluidic channel by balancing the electroosmotic flow and the electrophoretic migration of the particle.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

Microfluidic devices for the analysis of apoptosis

Apoptosis is the outcome of a metabolic cascade that results in cell death in a controlled manner. Due to its important role in maintaining balance in organisms, in mechanisms of diseases, and tissue homeostasis, apoptosis is of great interest in the emerging fields of systems biology. Research into cell death regulation and efforts to model apoptosis processes have become powerful drivers for new technologies to acquire ever more comprehensive information from cells and cell populations. The microfluidic technology promises to integrate and miniaturize many bioanalytical processes, which offers an alternative platform for the analysis of apoptosis. This review aims to highlight the recent developments of microfluidic devices in measuring the hallmarks as well as the dynamic process of cellular apoptosis. The potential capability and an outlook of microfluidic devices for the study of apoptosis are addressed.

Chemical and biological applications of digital-microfluidic devices

The advent of digital microfluidic lab-on-a-chip (LoC) technology offers a platform for developing diagnostic applications with the advantages of portability, reduction of the volumes of the sample and reagents, faster analysis times, increased automation, low power consumption, compatibility with mass manufacturing, and high throughput. Moreover, digital microfluidics is being applied in other areas such as airborne chemical detection, DNA sequencing by synthesis, and tissue engineering. In most diagnostic and chemical-detection applications, a key challenge is the preparation of the analyte for presentation to the on-chip detection system. Thus, in diagnostics, raw physiological samples must be introduced onto the chip and then further processed by lysing blood cells and extracting DNA. For massively parallel DNA sequencing, sample preparation can be performed off chip, but the synthesis steps must be performed in a sequential on-chip format by automated control of buffers and nucleotides to extend the read lengths of DNA fragments. In airborne particulate-sampling applications, the sample collection from an air stream must be integrated into the LoC analytical component, which requires a collection droplet to scan an exposed impacted surface after its introduction into a closed analytical section. Finally, in tissue-engineering applications, the challenge for LoC technology is to build high-resolution (less than 10 microns) 3D tissue constructs with embedded cells and growth factors by manipulating and maintaining live cells in the chip platform. This article discusses these applications and their implementation in digital-microfluidic LoC platforms. © 2007 IEEE.

Non-linear mechanical behavior of the elastomer polydimethylsiloxane (PDMS) used in the manufacture of microfluidic devices

Polydimethylsiloxane (PDMS) is the elastomer of choice to create a variety of microfluidic devices by soft lithography techniques (eg., [1], [2], [3], [4]). Accurate and reliable design, manufacture, and operation of microfluidic devices made from PDMS, require a detailed characterization of the deformation and failure behavior of the material. This paper discusses progress in a recently-initiated research project towards this goal. We have conducted large-deformation tension and compression experiments on traditional macroscale specimens, as well as microscale tension experiments on thin-film (≈ 50µm thickness) specimens of PDMS with varying ratios of monomer:curing agent (5:1, 10:1, 20:1). We find that the stress-stretch response of these materials shows significant variability, even for nominally identically prepared specimens. A non-linear, large-deformation rubber-elasticity model [5], [6] is applied to represent the behavior of PDMS. The constitutive model has been implemented in a finite-element program [7] to aid the design of microfluidic devices made from this material. As a first attempt towards the goal of estimating the non-linear material parameters for PDMS from indentation experiments, we have conducted micro-indentation experiments using a spherical indenter-tip, and carried out corresponding numerical simulations to verify how well the numerically-predicted P(load-h(depth of indentation) curves compare with the corresponding experimental measurements. The results are encouraging, and show the possibility of estimating the material parameters for PDMS from relatively simple micro-indentation experiments, and corresponding numerical simulations.

Simple telemedicine for developing regions: Camera phones and paper-based microfluidic devices for real-time, off-site diagnosis

This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial urine. The combination of patterned paper, a portable method for obtaining digital images, and a method for exchanging results of the assays with off-site diagnosticians offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients (e.g., in the developing world, in emergency management, and during field operations by the military) to obtain diagnostic information that might be obtained more effectively by less valuable personnel.

Continuous Formation and Separation of Calcium-Alginate Capsules Containing Viable Mammalian Cells in Microfluidic Devices

Microfluidic devices can be used for many applications, including the formation of well-controlled emulsions. In this study, the capability to continuously create monodisperse droplets in a microfluidic device was used to form calcium-alginate capsules.Calcium-alginate capsules have many potential uses, such as immunoisolation of cells and microencapsulation of active drug ingredients or bitter agents in food or beverage products. The gelation of calcium-alginate capsules is achieved by crosslinking sodiumalginate with calcium ions. Calcium ions dissociated from calcium carbonate due to diffusion of acetic acid from a sunflower oil phase into an aqueous droplet containing sodium-alginate and calcium carbonate. After gelation, the capsules were separated from the continuous oil phase into an aqueous solution for use in biological applications. Typically, capsules are separated bycentrifugation, which can damage both the capsules and the encapsulated material. A passive method achieves separation without exposing the encapsulated material or the capsules to large mechanical forces, thereby preventing damage. To achieve passiveseparation, the use of a microfluidic device with opposing channel wa hydrophobicity was used to stabilize co-laminar flow of im of hydrophobicity is accomplished by defining one length of the channel with a hydrogel. The chosen hydrogel was poly (ethylene glycol) diacrylate, which adheres to the glass surface through the use of self-assembled monolayer of 3-(trichlorosilyl)-propyl methacrylate. Due to the difference in surface energy within the channel, the aqueous stream is stabilized near a hydrogel and the oil stream is stabilized near the thiolene based optical adhesive defining the opposing length of the channel. Passive separation with co-laminar flow has shown success in continuously separating calcium-alginatecapsules from an oil phase into an aqueous phase. In addition to successful formation and separation of calcium alginate capsules,encapsulation of Latex micro-beads and viable mammalian cells has been achieved. The viability of encapsulated mammalian cells was determined using a live/dead stain. The co-laminar flow device has also been demonstrated as a means of separating liquid-liquidemulsions.

EFFECTS OF ION LIMITING CONDITIONS ON THE BEHAVIOR OF MICROFLUIDIC DEVICES

Finite numbers of ions are present in microfluidic devices. This leads to ion limiting effects in microfluidic channels and electrode surfaces. These effects include electrode surface changes and ion concentration gradient formation across microfluidic channels, and can influence microfluidic device behavior. A literature survey on the use of electrochemical analysis techniques in micro- and nanofluidic devices was carried out, which puts into perspective the importance of electrode surface changes with regards to analytical microfluidic applications. Surface changes in Pt wire electrodes under various physiological buffer and electric field conditions were investigated using cyclic voltammetry (CV), SEM-EDS and XPS. Effects of surface changes on electrochemical analysis performance of Pt wire and thin film electrodes were investigated. Electrode surfaces were subjected to varying phosphate buffer and electric field conditions, and their CV performance was monitored. Electrode surfaces were also studied with SEM-EDS. Two studies of ion concentration gradient formation in microfluidic channels were conducted. In the first, concentration gradients of H+ and OH- ions generated on electrode surfaces were found to cause significant pH decreases in certain buffer and electric field conditions, which was also found to play a key role in iDEP manipulation of proteins. The role of electrode surface reactions in this case shows the importance of understanding electrode surface changes in microfluidic devices. In the second study of ion concentration gradient formation, Cl- ion concentration gradient formation was attempted to be quantified upon electric field application across a KCl solution. Electrokinetic transport of the Cl- indicating fluorophore MQAE contributed significantly to the fluorescence microscopy signals collected, complicating Cl- quantification as a function of position and time. It was shown that a dielectric coating on electrode surfaces is effective at preventing MQAE electrokinetic transport.

Microfabrication processes for microfluidic devices on a single laser Workstation: direct writing lithography on SU-8, laser ablation on polymers and mask manufacturing

We demonstrate the capability of a laser micromachining workstation for cost-effective manufacturing of a variety of microfluidic devices, including SU-8 microchannels on silicon wafers and 3D complex structures made on polyimide Kapton® or poly carbonate (PC). The workstation combines a KrF excimer laser at 248 nm and a Nd3+:YVO4 DPSS with a frequency tripled at 355 nm with a lens magnification 10X, both lasers working at a pulsed regime with nanoseconds (ns) pulse duration. Workstation also includes a high-resolution motorized XYZ-tilt axis (~ 1 um / axis) and a Through The Lens (TTL) imaging system for a high accurate positioning over a 120 x 120 mm working area. We have surveyed different fabrication techniques: direct writing lithography,mask manufacturing for contact lithography and polymer laser ablation for complex 3D devices, achieving width channels down to 13μ m on 50μ m SU-8 thickness using direct writing lithography, and width channels of 40 μm for polyimide on SiO2 plate. Finally, we have tested the use of some devices for capillary chips measuring the flow speed for liquids with different viscosities. As a result, we have characterized the presence of liquid in the channel by interferometric microscopy.