CD4+ T-cell counting device for monitoring HIV/AIDS in resource limited settings

Anurag Tripathi
Graduate Student
Prof. Nikos Chronis Group
Mechanical Engineering

Our aim is to develop an inexpensive diagnostic device for the point-of-care (POC) monitoring of HIV/AIDS in the resource limited settings of the world. CD4+ T cell (a type of white blood cell) count in human whole blood has been used conventionally as the metric for proliferation of HIV in an infected individual and its progression into AIDS. We have been working towards developing a device which can perform CD4+ T-cell counting for HIV/AIDS monitoring. Towards this end, we have developed a cell trapping biochip for capturing human white blood cells (WBCs). Our preliminary findings indicate that this biochip with a novel 3D trapping architecture enables to obtain a high (> 90%) trapping efficiency of WBCs. We have also developed a high numerical aperture, spherical microlens array for on-chip imaging of micron sized objects. Employing these microlenses, we have demonstrated for the first time, use of microlenses for direct image formation on an inexpensive imaging sensor without the use of any intermediate optics. High NA (~0.5) microlenses were able to resolve 1µm resolution patterns comparable to the performance of conventional microscope objectives. Subsequently, we aim to integrate the cell trapping biochip with the microlens array to obtain a standalone benchtop device for HIV/AIDS monitoring.

Coupling push-pull sampling to multi-phase microfluidic devices for high spatial and temporal resolution monitoring of neurotransmitters

Thomas R. Slaney
Graduate Student
Prof. Robert T. Kennedy Group
Department of Chemistry

Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis.  Because of the low flow rates used (50 nL/min) it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples.  High temporal resolution also requires control of Taylor dispersion during sampling.  To meet these challenges, low-flow push-pull perfusion was coupled with segmented flow to achieve sampling at 200 ms temporal resolution at < 50 nL/min flow rates.  To demonstrate the utility and practicality of this method, it was used to monitor L-glutamate in the striatum of anesthetized rats at 7 s temporal resolution.  Up to 500 samples of 6 nL each were segmented by an immiscible oil and collected in a capillary tube. The samples were assayed for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay.  Fluorescence of the resulting plugs was monitored downstream.  Microinjection of 70 mM potassium in physiological buffered saline evoked L-glutamate concentration transients that had an average maxima of 4.2 ± 0.8 μM (n = 18 in 5 animals) and rise times of 22 ± 2 s.  These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.

Versatile Surface Immobilization Strategies using Reactive Coatings

Dr. Joerg Lahann
Department of Chemical Engineering
University of Michigan

Our improved understanding of molecular biology, microfabrication, and materials chemistry has stimulated crossfertilization of chemistry, biotechnology and materials engineering. In my presentation, I will discuss current advances in the design of multifunctional coatings including under research in the Lahann group. These reactive coatings with one or multiple functions can be synthesized by chemical vapor deposition (CVD) polymerization as well as CVD co-polymerization and may find use in a range of different biomedical applications.

Microfluidics device for in vivo imaging of neural response in small organisms

Mostafa Ghannad-Rezaie
Graduate Student
Prof. Nikos Chronis Group
Mechanical Engineering

Axons of most neurons have an intrinsic capacity to regenerate in response to injury. The regeneration is a complex mechanism regulated though diverse molecular pathways. Despite a few decades of studies still we have a limited understanding of the crucial events governing the axonal regeneration following an injury mainly because of technical difficulty associated with long-term time-lapse imaging and analysis. Here we introduce a simple microfluidics solution to study the neural response in larva Drosophila.

Microfluidic Devices for Spheroid Culture of Embryonic Stem Cells

Dr. Ji Yoon Kang
NanoBio Research Center
Korea Institute of Science and Technology

Embryonic stem (ES) cells are considered as a promising source for cell therapies or cell-based drug screening. However, a great number of biological experiments should be performed to study ES cell differentiation under controlled microenvironment. Lab-on-a-chips are advantageous in parallel massive experiments with small volume of reagents and are also beneficial to culturing cells under defined condition, i.e., serially diluted concentrations of chemicals. We developed microfluidic devices to generate and culture ES cell spheroids to observe its differentiation. The differentiation of ES cells usually begins with the aggregation of ES cells to form embryonic body (EB) and chemicals are applied to EB’s for differentiation. Conventional aggregation of ES cells for EB formation has been performed by hanging drop method or suspension culture; however the size of EB is irregular and it sometimes degrades the reliability of experimental results. We encapsulated ES cells in monodisperse alginate beads using droplet-generation microfluidic chips and the deviation of EB size was decreased. The neuronal differentiation of ES cells in alginate bead was tested in bead trap chip by retinoic acid treatment. Each bead was cultured in a separate chamber for more than a week and neuronal cells were clearly observed by confocal microscope in the beads. This model experiments suggest that this chip can be used as a microfluidic platform to study the effect of chemicals on ES cell differentiation in embryoid body.

Micro-hydrodynamics of deformable objects: application to bacterial swimming

Nobuhiko Watari
Graduate Student
Prof. Ron Larson Group
Department of Applied Physics

Multi-flagellated bacteria, such as Escherichia coli, often have flagella attached at random locations to the cell body. To study the effect of the number of flagella and the geometric arrangement of them to the swimming efficiency, we develop a simulation method using a bead-spring model to account for the hydrodynamic and the mechanical interactions between multiple flagella and the cell body. First, a modeled bacterium is constructed using beads, which represent the hydrodynamic drag centers of the geometric elements of the bacterium.  These beads are bonded by 1) a spring potential, 2) a bending potential, and 3) a torsional potential to adjacent beads. This modeled bacterium swims by rotating the flagella with constant torques at the bases of them. We have found that for modeled bacteria with two flagella, the swimming speed varies by 30% depending on the position of the base of the flagellum along the cell body, which affects the tightness of the bundling.   In general, by changing the geometric arrangement and the number of flagella, our simulation enables us to determine the optimal designing of a flagellated micro-swimmer.

Nanoelectrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Structural Analysis of Nucleic Acids and Phosphorylated Peptides

Hangtian Song
Graduate Student
Prof. Kicki Hakansson Group
Department of Chemistry

Electrospray ionization mass spectrometry (ESI-MS) is a key technique for identification and structural analysis of biomolecules such as peptides, proteins and nucleic acids. Nano-ESI, compared to regular ESI, introduces benefits such as less sample consumption, higher degree of preservation of solution-phase structure, and more tolerance for impurities. Here we show that a commercial chip-based nano-ESI system interfaced with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer demonstrates the ability to obtain structural information for nucleic acid-small molecule complexes with several tandem mass spectrometric (MS/MS) techniques. MS/MS involves gas-phase activation and fragmentation of molecular ions of interest (precursor ions). Structural information is obtained from the observed fragment ions and known fragmentation patterns for particular classes of precursor ions. Utilization of MS/MS techniques requires additional time than single-stage MS and may be challenging to synchronize with on-line separation techniques, such as high performance liquid chromatography (HPLC). Here we also demonstrate coupling of MS/MS with segmented-flow sample collection, which preserves chromatographic resolution. Specifically, segmented flow was coupled with a novel MS/MS technique, negative ion electron capture dissociation (niECD), which is valuable for phosphopeptide analysis but more time consuming than other MS/MS techniques.

Integrated Bio-Microfluidic Circuits

Dr. Shuichi Takayama
Professor
Department of Biomedical Engineering
University of Michigan

Computers are made from integrated electronic circuits.  A significant part of our bodies consist of fluidic circuits constructed from cells and biochemicals.  This presentation will give an overview of efforts in our laboratory to recreate aspects of the biological microfluidic systems.  To recreate spatio-temporal patterns of chemical and fluid mechanical environments that cells may experience in the body.  The methods close the “gap” between conventional cellular studies performed in static dish cultures to provide biological information otherwise difficult to obtain.  Some of the fluidic circuits also aim to close the “gap” between microfluidic circuits and microelectronic circuits in terms of versatility and real-time, on-chip, self-regulatory capabilities.  Specific technological topics that will be discussed include development of compartmentalized microfluidic devices, self-regulating microfluidic circuits, micropatterning using aqueous two-phase systems, and tunable nanochannels.  Biological topics to be presented include application of the technologies to in vitro fertilization, engineering of stem cell niches, analysis of cell signaling, and single molecule DNA and chromatin analysis.

Developing Microfluidic Mixers to Study Protein Folding

Dr. Lisa Lapidus
Associate Professor
Department of Physics
Michigan State University

Protein folding is an enormously difficult problem to solve in part because there is such a wide range of relevant timescales.  Currently, there is a large gap between the times accessible to most molecular dynamics simulations of folding proteins and the standard experimental method of stopped-flow mixing to prompt protein folding.  To fill this gap my lab has developed microfluidic mixers that eliminate turbulence and mix quickly.  We currently have the fastest mixer in the world, which can mix solutions in about 4 microseconds, almost 1000 times faster than conventional mixers.  I will describe the fabrication of various mixers developed in my lab along with the optical instruments that use them.

Controlling Translocation through Nanopores with Bio-Inspired, Fluid Walls

Dr. Michael Mayer
Associate Professor
Department of Chemical Engineering and Department of Biomedical Engineering
University of Michigan

Synthetic nanopores are emerging as promising tools for fundamental and applied studies of individual biomolecules in high throughput; their performance as sensors can, however, currently not match that of sensory nanopores in biology such as ion channel proteins.  This talk introduces the concept of synthetic nanopores with fluid lipid walls in an attempt to shorten this gap.  The inspiration for this design stems from lipid-coated nanopores in olfactory sensilla of insect antennae.  We demonstrate that multifunctional coatings of fluid lipids confer unprecedented functionality to nanopore-based sensors and address currently unmet challenges of single protein investigations with nanopores.  For instance, bilayer coatings made it possible to fine-tune and actuate pore diameters with sub-nanometer precision.  Incorporation of lipid-anchored ligands conferred specificity and slowed down translocation of targeted proteins sufficiently to resolve complete translocation events of individual proteins.  The biocompatible, fluid nature of bilayer coatings prevented pore clogging and enabled the first translocation analysis of amyloid-beta oligomers.  Bilayer coatings also eliminated non-specific binding and facilitated the combined, quantitative analysis of translocation time, volume, charge, and ligand affinity of single proteins.

CVD-based Polymer Substrates for Spatially-resolved Analysis of Protein Binding by Imaging Ellipsometry

Aftin Ross
Lahann Lab
Department of Chemical Engineering

Biomolecular interactions with synthetic surfaces are important in diverse biomedical fields including medical device implants, microfluidic sensors, marine fouling, and tissue engineering. In the past, chemical vapor deposition (CVD) polymerization has been used to fabricate thin polymer films with robust mechanical and physical properties. Functional groups on CVD coatings serve as anchors for the immobilization of biomolecules and thus the affinity of surfaces for various proteins, antibodies, or antigens can be modulated. As a result, CVD films are well suited for use as protein/antigen-antibody sensors or in controlling the cellular microenvironment for microfluidic cell culture. However, for many of applications, it would be beneficial to understand kinetics, or time based interactions, of CVD surfaces with biomolecules as well as the extent of these interactions. Therefore a cascade of surface modifications on CVD coatings was explored using a microfluidic technique known as surface plasmon resonance enhanced ellipsometry (SPREE). CVD films are well suited for SPREE studies because in addition to their biofunctionality, they are easily attached to gold which serves as a substrate for SPREE assays. In this work, we exploit protein-surface interactions in order to demonstrate the feasibility of thin film CVD surfaces, in particular a functionalized [2.2]paracyclophanes (PPXCOC2F5), as a spatially-resolved binding array for SPREE studies. Additional analysis techniques include Fourier transform infrared spectroscopy (FTIR), electrical impedance spectroscopy (EIS), imaging ellipsometry, and fluorescence microscopy. FTIR confirmed the presence of the thin film and EIS indicated that CVD films were pin hole free. Results from imaging ellipsometry verified that films of varying thickness were created which then allowed the impact of film thickness on protein sensing to be assessed. SPREE and fluorescence microscopy findings demonstrated that CVD films acted as antigen-antibody sensors and that SPREE sensitivity is impacted by increasing film thickness.

Using interstitial fluid flow and tensile cyclic strain to study the fibroblast to myofibroblast transition in vitro

Peter Galie
Prof. Jan Stegemann Group
Department of Biomedical Engineering

The fibroblast to myofibroblast transition is a crucial event in the response to myocardial infarction. The myofibroblast phenotype is characterized by increased cell contractility, expression of alpha-smooth muscle actin (?-SMA), and extracellular matrix deposition that facilitates the formation of a fibrotic scar in the wall of the heart. Understanding the mechanisms of this transition has implications for therapies aimed at preventing the eventual progression to heart failure. Early work indicated that mechanical stress may be an important determinant of fibroblast/myofibroblast phenotype. In order to investigate the effect of mechanical stimuli, a mesoscale PDMS substrate was constructed to apply both interstitial fluid flow and cyclical tensile strain to fibroblast-seeded collagen hydrogels. A linear biphasic finite element model can predict the stress generated within the hydrogel during mechanical stimulation within the PDMS substrate. Results indicate that interstitial fluid flow and cyclic tensile strain produce opposing responses from the fibroblasts seeded in collagen gels. Several antibodies have been used in concert with the PDMS apparatus to discern mechanisms for this cellular mechanotransduction. Current research is investigating how the myofibroblast phenotype affects the dedifferentiation of adult cardiomyocytes.