David W.M. Marr - US grants

A highly innovative polymer laboratory class is created to accompany an increasingly popular interdisciplinary polymer science lecture class. Carefully considered and selected state-of-the-art instrumentation is acquired and used in conjunction with "just in time" learning under the premise of the student's being faced with realistic industrial situations whereby specific technical questions have to be addressed within a limited period of time. Five separate experiments emphasizing and reinforcing the material presented in lecture are performed. The laboratory class is team taught by one faculty member from Chemical Engineering and one from the Chemistry Department; heavy contact hours between the student and these faculty in an intimate laboratory setting are an indispensable part of the course. *

CTS-9734136 Marr, David W. M. Colorado School of Mines Structure at small length scale arises as the material microscopic components interact and assemble themselves into mesoscopic-sized morphologies. This project will investigate the link between molecular interactions and the properties of interfaces that develop during morphology evolution. A combination of dense colloidal model systems probed by new optical techniques that can manipulate such systems, and statistical mechanical modeling methods will be applied. A new experimental device will be constructed that uses a rapidly scanning optical trap to build arbitrary optical potential wells. The technique of confocal microscopy which can characterize, in situ, the relatively dense colloidal structures will be developed. The study of nucleation in systems using nuclei of various structure and the templated growth of colloidal crystals will be two areas of focus. The education goal is to bring the physical insights obtained in this study to the classroom at both undergraduate and graduate levels by developing new courses and adding molecular level material to current coursework. In addition, new pedagogical techniques including the World Wide Web, browsers used to access the web, as well as state-of-the-art computer languages will be used to enhance the educational environment. Understanding the link between microscopic interactions in colloidal systems and macroscopic properties is an essential step in the synthesis of new materials with specifically designed microscale morphologies. ***

Abstract
CTS-0097841
David Marr, Colorado School of Mines

The PI proposes to develop colloid-based flow control devices capable of sensing and actuating using optical trapping techniques. Micro-sized check valve, directional valve, and colloid pump are to be built based on single colloidal particles to effect flow control. These devices will have broad applications in biological and engineering systems.

We have recently developed a novel and general means of ordering colloidal particles into three-dimensional crystals rapidly and reversibly using electric fields. The ordering mechanism appears to rely on a combination of induced dipole interactions and hydrodynamic flows that push particles together, creating an effective attraction. Because these interactions can be moderated with a combination of applied field properties and confining geometry, their magnitude can be greatly tuned and used to crystallize particles in a wide variety of size ranges. Our preliminary investigations have focused on colloids ranging in size from 250 nm down to 50 nm in radius, all of which can be reversibly crystallized through application of the external field. We have also shown that nucleation of these colloidal crystals can be spatially controlled solely through selective application of the electric field. The goal of the work proposed here is to extend these preliminary investigations into smaller length scales throughout the nanoscale range. We intend to take advantage of this for the reversible and spatially addressable crystallization of nanoscale particles. In addition to the creation of a display technology with inexpensive materials, we envision the development of switchable photonic band gap materials based upon the reversible ordering of nanocolloidal-sized materials. The award has been funded by the Thermal Transport and Thermal Processing Program and the Particulate and Multiphase Processes Program of the Chemical and Transport Systems Division.

This award is for a collaboration with DBI-0454763. It supports the development of a novel multiphoton microscope that will be capable of measuring biomolecular dynamics over 15 orders of magnitude in time. The microscope will use microfluidics technology to provide microsecond time-scale mixing, and low (ml/hour) sample consumption rates. Microsecond to second time scale delays will be achieved by using a microscope to probe the sample flow at increasing distances (corresponding to increasing time) along the outlet channel. The optical system will implement three pulse echo peak shift spectroscopy (3PEPS), a four-wave mixing method that has proven useful for measuring the spectrum of fluctuations of cofactor-containing proteins. The microscope will initially be employed in two lines of work: 1) studying the evolution of flexibility and heterogeneity during protein folding and 2) characterizing the dynamics of intermediate species in the photocycle of photoactive yellow protein.

Direct measurements of the molecular motions of biomolecules over a broad time range is crucial to furthering a microscopic understanding of biology. To fully appreciate the relation between molecular motions and biochemical events, the time scale over which molecular dynamics must be recorded is enormous - from femtoseconds (10-15 seconds) to seconds or even minutes. This collaborative work brings together the expertise of an optical physicist, chemical engineer, and biophysicist, to develop instrumentation that will advance knowledge in biomolecular dynamics. This new instrumentation will stimulate broader application of powerful femtosecond nonlinear optical techniques to biochemical studies, and will further develop technologies with great promise in biological studies: multiphoton microscopy and microfluidics. This research will benefit interdisciplinary biophysics education at the undergraduate and graduate levels by providing opportunities for students to work in multidisciplinary teams including molecular biologists, chemical engineers, and optical physicists.

Identifying a certain cell type based on a specific signature, separating cell mixtures according to cellular differences, and studying changes in a specific signature with cell environments are important techniques in biological research. While various biochemical markers of cells have been extensively used for decades, biomechanical properties such as cell stiffness are increasingly recognized as an important indicator of cell type and physiological state. The mechanical properties of cells are defined by the membrane, cytoskeleton and the volume of the cell, and are likely associated with basic characteristics including type, growth, stage of differentiation, and response to the environment. Existing mechanical characterization tools, however, can only examine a few cells at a time, severely limiting their utility and application due to the low throughput associated with the sequential isolation and probing of individual cells.
Here, a high-throughput method will be developed, where optical-based mechanical ?stretching? forces are applied to cells in microfluidic devices. By the end of this 3-year project, a device capable of the rapid measurement of cell mechanical properties will be built and tested on bovine blood cells, vascular cells, and human HeLa cells. A number of broader impacts are expected including providing new research opportunities for undergraduate students, new teaching opportunities at the high-school level, and new recruitment efforts for underrepresented groups within the state of Colorado.

Project Summary: We introduce a unique microfluidic-based approach for the high-throughput non-destructive assaying of cells without the need for specific labels or reagents. Based on measurement of both static and dynamic cell mechanical properties using applied optical forces, we will apply this technique (known as optical stretching) in a high-speed high-throughput manner. To date, optical stretching has been used only on small cell numbers; however, high- intensity, microscale laser sources and the integration of these within dynamic microfluidic systems has enabled our proposed approach. In this, fully integrated optical-based sensors and mechanical stretchers will be used to identify and, upon demand, isolate single cells. Once identified, such targeted cells can then be transported on-chip to culture chambers within the device or for dispensing into standard bio-laboratory instrumentation for off-chip analysis. Though there is broad need, our proposed technology will be tested and developed using malaria parasite infected red blood cells as the target cell. This work will be done in collaboration with the Laboratory of Malaria and Vector Research at the NIAID. Our aims include: Aim 1: Mechanical Property Detection and Interpretation. We will employ optical manipulation methods integrated within microfluidic systems for label-free, non-destructive cell mechanical property measurement. Modeling approaches will be developed for both interpretation of applied force/deformation experimental data and for device design. Here, malaria-infected red blood cells will provide a good model target since cell stiffness changes dramatically during parasite development. Demonstrating greatly simplified device designs and associated ease-of-use, we will install an instrument in an active NIH laboratory. Aim 2: Optical Manipulation for Cell Identification and Isolation. We will integrate optical methods within microfluidic systems for single cell detection and manipulation. Here, methods for both on-chip cell isolation and off-chip isolation will be developed and used to improve our installed NIH protototype. Aim 3: High Throughput Mechanical Testing. To achieve high-throughputs, modified microfluidic and faster detection techniques will be required. In this phase, the coupling of hydrodynamic and optical forces will be explored to improve device performance. In addition, time-varying optical forces will be employed to identify optimal signal response and dynamic physical properties.

DESCRIPTION (provided by applicant): Ischemic strokes can be treated with either chemical or mechanical means, each with advantages and disadvantages. Tissue plasminogen activator (tPA), a common clot buster, has been used to treat thrombotic clots but can lead to excessive bleeding and must be used soon after symptoms first occur. Mechanical methods can restore blood flow quickly but are invasive and can leave residual prothrombotic material on vessel walls, increasing risk for secondary stroke. To address these drawbacks, we propose a targeted delivery approach performed through an injectable colloidal solution controlled by an external magnetic field. This non-invasive approach combines pharmacological and mechanical methods for clot removal. Here, individual particles in solution are injected into the blood and, upon application of a magnetic field, self-assemble into small microdevices capable of targeting fibrinolytic agents and mechanically attacking a clot in the absence of catheters. As both microdevice assembly and driving forces are provided by the external field, once the procedure is finished, devices self- disassemble into small building blocks removable by the body via phagocytosis. We note that, as the approach is microscale in nature, it can be tuned to more carefully remove any prothrombotic residual clot that can arise in mechanical thrombectomies. Our aims include: Specific Aim 1: Determine the rate at which colloidal-based devices mechanically remove clots. We will investigate clot removal rate by mechanical disruption as a function of operating parameters such as microdevice size and spin-rate within microfluidic vascular mimics. Specific Aim 2: Determine the effectiveness with which fibrinolytic-modified colloidal microdevices can be used to enhance clot removal. Here, we will synthesize tPA-modified magnetic beads and demonstrate their use as fibrinolytic agents within microfluidic vascular mimics. We expect direct coupling of tPA to enhance dissolution rates over mechanical disruption alone. Aim 3: Demonstrate device assembly and targeting within in vivo environments. With a well-established animal stroke model we will demonstrate the delivery, assembly, and targeting of magnetic assemblies to the site of vascular occlusion. Imaged with available small animal MRI facilities, these studies will provide the necessary proof-of-principle for further investigations.

Project Summary: In small vessel stroke (SVS), which accounts for 20% of ischemic strokes, tissue plasminogen activator (tPA) is ineffective because it can take a prohibitively long time to diffuse to the clot, and catheter-based thrombectomy devices cannot access small vessels. Moreover, treatment associated hemorrhaging limits tPA use to within a few hours of the onset of symptoms for all ischemic strokes. As a result, there is an urgent need for strategies that overcome these limitations, particularly in SVS, while reducing the risks associated with tPA. Building on a successful previous work, a drug delivery strategy is proposed that can selectively target small artery occlusions and deliver mechanical force to accelerate thrombolysis. The objective of this proposal is to investigate and test within realistic models an approach where injected, dispersed magnetic beads are assembled into blood cell sized microwheels (µwheels) capable of targeting occlusive clots located in small vessels and lysing them with a combination of mechanical and biochemical action. The central hypothesis is that µwheels can (i) target occluded small arteries by exploiting the low flow regions at the entrance of these vessels, (ii) achieve reperfusion at rates an order-of-magnitude faster than soluble tPA, and (iii) improve outcomes in murine models of stroke. This hypothesis will be tested with the following specific aims: Aim 1. Identify magnetic field conditions for µwheels targeting of occlusions. µWheels will be assembled in flowing blood and directed to occluded channels or vessels. Microfluidic, zebrafish, and 3D human cerebrovascular models will be used to test the assembly and targeting. Aim 2. Determine rates for thrombolysis of occlusive thrombi using tPA functionalized µwheels. It is postulated that tPA functionalized µwheels can dissolve fibrin- and platelet-rich clots within microfluidic models and achieve reperfusion in zebrafish and 3D human cerebrovascular models, at rates significantly faster than soluble tPA. Aim 3. Measure the functional benefit of µwheel thrombolysis in vivo. In comparison to soluble tPA, µwheel mediated thrombolysis will improve safety, motor, and neurological outcomes in murine stroke models and can be visualized using high-resolution MRI and micro-CT. In Aims 1 and 2 the expected outcomes are identifying the operating conditions for µwheel assembly, targeting, and fibrinolysis that provide faster reperfusion compared to tPA and can be scaled-up to human-size vascular networks. In Aim 3, it will be shown that µwheel thrombolysis is a superior strategy to systemic administration of tPA in terms of neurobehavioral outcomes in a stroke model and can be imaged in vivo. This approach is significant because it could lead to the development of a more rapid and less invasive strategy for alleviating ischemia than methods currently available. This approach is innovative because of the use of external magnetic fields to propel fibrinolytic microdevices to the sites of occlusion and provide mechanical action to accelerate reperfusion time compared to systemic administration of tPA.

Project Summary: In cystic fibrosis (CF), treatment is difficult because chronic lung infections lead to biofilm formation and drug-resistant bacterial strains prevent antibiotics from working effectively. Antibiotic approaches specifically designed to address infections in the lung include inhaled antibiotics; however, resistant strains are a significant challenge. For delivery, such inhaled drugs must be formulated within a specified size range. Too large and they do not remain suspended to reach deep within the lungs. Too small and they remain in the air and are simply exhaled without embedding. Our scientific premise is that individual magnetic particles of this optimal size range can be inhaled into the lungs and subsequently assembled in place in the form of wheel-like assemblies, or µwheels, to travel deep down lung pathways and disrupt mucus layers to enhance drug-induced biofilm removal. As both µwheel assembly and driving forces are provided by an external magnetic field, once the procedure is finished, devices ?self- disassemble? into small building blocks removable by the body's natural mechanism for removal of dust and other foreign particles in the mucus lining. Our aims include: Aim 1: Identify applied magnetic field conditions that promote µwheel-enhanced biofilm degradation. We will study model P. aeruginosa and CF patient-derived biofilms, with and without artificial sputum, and attach antibiotics or dispersal agents onto the magnetic particle surface and within tortuous microenvironments. We will also use nanoparticle-decorated µwheels to perforate and penetrate the film and test with antibiotic/dispersal agent in solution. Aim 2: Determine conditions that support airborne delivery and transport of µwheels in 3D environments. We will demonstrate airborne delivery of µwheels and translation within 3D models of patient respiratory systems, with and without artificial sputum.

The field of microrobotics seeks to develop very small machines capable of performing tasks that cannot currently be accomplished with traditional instruments. Often targeted for biomedical applications within the body, microrobots (microbots) could be used to deliver chemotherapeutic drugs to specific locations to treat cancer or to break up blockages within blood vessels as in the case of stroke or in heart diseases. Challenges remain however both in fabricating microbots and powering them, especially when biocompatible materials are desired. This award will study and test the use of soft liquid microbots, created with a magnetic shell that allows directed rolling with application of an external magnetic field. Fabricated using emulsion-based techniques, this approach allows for easy synthesis, ready control, enhanced biocompatibility, and efficient drug delivery. The motion of these soft microbots over surfaces is different from their rigid counterparts. This award will investigate the traction and movement of soft microbots over a variety of hard and soft surfaces under the applied magnetic fields to enhance and optimize their movement for targeted applications.

Microbot locomotion is challenging because of the reversible nature of microscale fluid flow, a limitation that can be overcome by breaking flow-field symmetry with a nearby surface. When translating along an interface, rolling microbots travel faster than sliding ones because of their significantly smaller friction coefficient. This phenomenon has been utilized with rotating wheel-shaped microbots for targeting occlusive clots located in small arteries, delivering drugs, and inducing lysis via a combination of mechanical and biochemical action. Recent studies, however, have used rigid microbots that roll inefficiently with significant slip and difficulty navigating confined environments. Addressing these drawbacks, this award proposes the use of soft microbots based on superparamagnetic Pickering emulsions. They will provide enhanced biocompatibility and large drug encapsulation capacity and can alter their shape to enhance traction by accommodating interactions with viscoelastic in vivo environments. Soft Pickering emulsion microbots will be fabricated and the nature of their transport on various surfaces and in various environments will be investigated. The ability of these soft microbots to transport and deliver drugs within various biomimetic environments will also be explored.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.