Dmitry A. Markov

Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry

Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kds and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

Engineering Challenges for Instrumenting and Controlling Integrated Organ-on-Chip Systems

The sophistication and success of recently reported microfabricated organs-on-chips and human organ constructs have made it possible to design scaled and interconnected organ systems that may significantly augment the current drug development pipeline and lead to advances in systems biology. Physiologically realistic live microHuman (μHu) and milliHuman (mHu) systems operating for weeks to months present exciting and important engineering challenges such as determining the appropriate size for each organ to ensure appropriate relative organ functional activity, achieving appropriate cell density, providing the requisite universal perfusion media, sensing the breadth of physiological responses, and maintaining stable control of the entire system, while maintaining fluid scaling that consists of ~5 mL for the mHu and ~5 μL for the μHu. We believe that successful mHu and μHu systems for drug development and systems biology will require low-volume microdevices that support chemical signaling, microfabricated pumps, valves and microformulators, automated optical microscopy, electrochemical sensors for rapid metabolic assessment, ion mobility-mass spectrometry for real-time molecular analysis, advanced bioinformatics, and machine learning algorithms for automated model inference and integrated electronic control. Toward this goal, we are building functional prototype components and are working toward top-down system integration.

Neurovascular unit on a chip: implications for translational applications

The blood-brain barrier (BBB) dynamically controls exchange between the brain and the body, but this interaction cannot be studied directly in the intact human brain or sufficiently represented by animal models. Most existing in vitro BBB models do not include neurons and glia with other BBB elements and do not adequately predict drug efficacy and toxicity. Under the National Institutes of Health Microtissue Initiative, we are developing a three-dimensional, multicompartment, organotypic microphysiological system representative of a neurovascular unit of the brain. The neurovascular unit system will serve as a model to study interactions between the central nervous system neurons and the cerebral spinal fluid (CSF) compartment, all coupled to a realistic blood-surrogate supply and venous return system that also incorporates circulating immune cells and the choroid plexus. Hence all three critical brain barriers will be recapitulated: blood-brain, brain-CSF, and blood-CSF. Primary and stem cell-derived human cells will interact with a variety of agents to produce critical chemical communications across the BBB and between brain regions. Cytomegalovirus, a common herpesvirus, will be used as an initial model of infections regulated by the BBB. This novel technological platform, which combines innovative microfluidics, cell culture, analytical instruments, bioinformatics, control theory, neuroscience, and drug discovery, will replicate chemical communication, molecular trafficking, and inflammation in the brain. The platform will enable targeted and clinically relevant nutritional and pharmacologic interventions for or prevention of such chronic diseases as obesity and acute injury such as stroke, and will uncover potential adverse effects of drugs. If successful, this project will produce clinically useful technologies and reveal new insights into how the brain receives, modifies, and is affected by drugs, other neurotropic agents, and diseases.

SiO2-coated porous anodic alumina membranes for high flow rate electroosmotic pumping

Electroosmotic pumping has been extensively used in lab-on-a-chip devices and micropumps for microelectronic cooling. High flow rate per unit area with a low applied voltage is a key performance requirement to achieve compact design and efficient operation. In this paper, we report work on using SiO2-coated porous anodic alumina membranes for high flow rate electroosmotic pumping under low applied voltages. High quality porous alumina membranes of controllable pore diameters in the range of 30‐100 nm and pore lengths of 60‐100 μm were fabricated by electrochemical anodization. The pores are straight, uniform and hexagonally close-packed with a high porosity of up to 50% of the total area. The inner surface of the pore was coated conformally with a thin layer (∼ 5n m) of SiO 2 to achieve a high zeta potential. The electroosmotic pumping performance of the fabricated anodic alumina membranes, coated and uncoated, was investigated using standard relevant aqueous electrolyte buffer solutions. The high zeta potential of the SiO2 coating increases the pumping flow rate even though the coating reduces the porosity of the membrane. Results show that nanostructured SiO2-coated porous anodic alumina membranes can provide a normalized flow rate of 0.125 ml min −1 V −1 cm −2 under a low effective applied voltage of 3 V. This compares favourably with other microporous materials such as glass frits.

Metabolic consequences of inflammatory disruption of the blood-brain barrier in an organ-on-chip model of the human neurovascular unit

BackgroundUnderstanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches—whether in vivo or in vitro—have often been only snapshots of this complex web of interactions.MethodsWe utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on blood-brain barrier function ex vivo and the metabolic consequences of these responses and repair mechanisms. In this study, we pair a novel dual-chamber, organ-on-chip microfluidic device, the NeuroVascular Unit, with small-volume cytokine detection and mass spectrometry analysis to investigate how the blood-brain barrier responds to two different but overlapping drivers of neuroinflammation, lipopolysaccharide and a cytokine cocktail of IL-1β, TNF-α, and MCP1,2.ResultsIn this study, we show that (1) during initial exposure to lipopolysaccharide, the blood-brain barrier is compromised as expected, with increased diffusion and reduced presence of tight junctions, but that over time, the barrier is capable of at least partial recovery; (2) a cytokine cocktail also contributes to a loss of barrier function; (3) from this time-dependent cytokine activation, metabolic signature profiles can be obtained for both the brain and vascular sides of the blood-brain barrier model; and (4) collectively, we can use metabolite analysis to identify critical pathways in inflammatory response.ConclusionsTaken together, these findings present new data that allow us to study the initial effects of inflammatory stimulation on blood-brain barrier disruption, cytokine activation, and metabolic pathway changes that drive the response and recovery of the barrier during continued inflammatory exposure.

Ultrasmall volume refractive index detection using microinterferometry

A microinterferometric backscatter detector (MIBD) has been developed to perform subnanoliter volume refractive index measurements using a simple, folded optical train based on the interaction of a laser beam and a fused silica capillary tube. Positional changes of the interference pattern extrema (fringes) allow for the determination of Δn at the 10−7 level, corresponding to 5.3 pmole or 0.48 ng of solute, when thermal noise is controlled at 8×10−3 °C. MIBD is relatively path-length insensitive for capillaries ranging in inner diameter from 75 to 775 μm, allowing a large range of detection volumes, from 350 pL to 40 nL, to be produced. A theoretical model of the microinterferometric backscatter detector has also been developed and evaluated and has been found to be in agreement with experimental data. This model indicates increased sensitivity of the instrument as the wavelength of the probe beam and the wall thickness of the capillary tube are reduced.

Micro-interferometric backscatter detection using a diode laser

Micro-interferometric backscatter detection (MIBD) is performed with a simple, folded optical train based on the interaction of a diode laser beam and a fused silica capillary tube allowing for refractive index (RI) determinations and detection of optically active molecules in small volumes. Side illumination of the capillary by a laser produces a 360 fan of scattered light that contains two sets of high contrast interference fringes. These light and dark spots are viewed on a flat plane in the direct backscatter configuration. Signal interrogation for polarimetry is based on quantifying the relative intensities (depth of modulation (DOM)) of adjacent high frequency (HF) interference fringes for polarimetry and relative fringe position for RI detection. Positional changes of the interference pattern extrema (fringes) allow for the determination of 1n at the 10 7 level or 5.3 pmol or 0.48 ng of solute. The MIBD-RI detection volume is just 5.0 nl. DOM changes allow for optical activity detection limits of 5.7 10 5 (mandelic acid, [a] 23 = 153, and D-glucose, [a] 25 = +52.5), and a 2 detection limit of 7.5 10 4 M (D-glucose) and 1.14 10 3 M (R-mandelic acid). The probe volume of MIBD-polarimetry was 38 nl, and within the probed volume at the limit of detection, about 28.7 pmol of mandelic acid or about 43.7 pmol of D-glucose is present. Furthermore, DOM (polarimetry signal) is unchanged when a non-optically active solute is interrogated by the MIBD-polarimeter. Finally, an optical model was derived and used to evaluate the advantages and pitfalls of using diode laser for MIBD. ©1999 Elsevier Science B.V. All rights reserved.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy.

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size‐matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright‐field and fluorescent side‐view images, and when combined with z‐sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five‐fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low‐light imaging modalities such as bioluminescence, or low abundance surface‐marker labelling.

Tape underlayment rotary-node (TURN) valves for simple on-chip microfluidic flow control

We describe a simple and reliable fabrication method for producing multiple, manually activated microfluidic control valves in polydimethylsiloxane (PDMS) devices. These screwdriver-actuated valves reside directly on the microfluidic chip and can provide both simple on/off operation as well as graded control of fluid flow. The fabrication procedure can be easily implemented in any soft lithography lab and requires only two specialized tools—a hot-glue gun and a machined brass mold. To facilitate use in multi-valve fluidic systems, the mold is designed to produce a linear tape that contains a series of plastic rotary nodes with small stainless steel machine screws that form individual valves which can be easily separated for applications when only single valves are required. The tape and its valves are placed on the surface of a partially cured thin PDMS microchannel device while the PDMS is still on the soft-lithographic master, with the master providing alignment marks for the tape. The tape is permanently affixed to the microchannel device by pouring an over-layer of PDMS, to form a full-thickness device with the tape as an enclosed underlayment. The advantages of these Tape Underlayment Rotary-Node (TURN) valves include parallel fabrication of multiple valves, low risk of damaging a microfluidic device during valve installation, high torque, elimination of stripped threads, the capabilities of TURN hydraulic actuators, and facile customization of TURN molds. We have utilized these valves to control microfluidic flow, to control the onset of molecular diffusion, and to manipulate channel connectivity. Practical applications of TURN valves include control of loading and chemokine release in chemotaxis assay devices, flow in microfluidic bioreactors, and channel connectivity in microfluidic devices intended to study competition and predator/prey relationships among microbes.