Aqueous Two-Phase System Assays to Improve the Drug Development Process

Josh White
Graduate Student
Takayama Lab
Biomedical Engineering

Since the 1990s, a decade for pharmaceutical companies that was marked by blockbuster drug development, there has been a significant decline in the creation of breakthrough medicines despite an increase in investments in R&D. This decline in productivity can be thought of in terms of reduced efficiency, the decline in number of drugs produced from a library of potential compounds, and reduced effectiveness, the inability to translate drug candidates into revenue-producing pharmaceuticals. Along the spectrum of drug development, potential compounds are selected based on initial screening against a library of biomarkers. After chemical characterization and optimization, compounds are then analyzed in vitro for on-target and off-target effects as well as to determine specific mechanisms of action. Drugs are finally tested in vivo in animal models and clinical trials. Aqueous two-phase system assays can improve biomarker discovery and identification by enabling high-throughput and multiplexed detection of proteins and also by creating more physiologically relevant in vitro cell culture to provide more robust early screening of compounds.

Fluid handling operations in droplet microreactors: optical tweezing, sorting, particle segregation, and label free sensing in picoliter volumes

Amar S. Basu
Assistant Professor
Electrical and Biomedical Engineering
Wayne State University

Multiphase microfluidics utilizes water-in-oil droplets as containers for biochemical reactions.  With nL-pL volumes, they provide a hundred to million-fold smaller reaction volumes than conventional microplates or single-phase microfluidic channels.  As modern biology evolves from studies of single genes and proteins to systematic, high-throughput studies involving of thousands of biomolecules, drop-based fluidics can provide compelling benefits to the fields of ‘-omics’ and drug discovery, including: dramatic cost reductions by conserving expensive reagents, high throughput due to lower diffusion times, and novel ways to query single cells in small volumes.  To be able to reap these benefits, however, one must be able to emulate typical benchtop fluid handling operations in the droplet format.  Multiphase fluidics can be considerably more complex than single-phase microfluidics as they involve coupled flow interactions between two immiscible phases, interfacial tension, Laplace pressures, shape deformation, and other phenomena acting in cohort with the standard flow profiles.  Rather than work against these phenomena, we exploit them in novel ways in order to perform key fluid handling operations.  Several examples will be discussed in this talk: 1) the formation of heterogeneous screening libraries using microfractionation-in-droplets (µFD); 2) Particle concentration using hydrodynamic microvortices and sedimentation in plug flow; 3) Optical droplet manipulation using optofluidic tweezers (OFT), a novel technique which uses laser-induced thermocapillary microvortices to trap droplets with µN forces (100,000X larger than traditional optical tweezers); 4) Sorting droplets by size using tensiophoresis, the cross-stream migration of droplets in interfacial tension (IFT) gradients; and 5) Detecting femtomoles of proteins in droplets using interfacial adsorption phenomena.  Computational modeling and experimental image velocimetry methods will also be discussed.

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Quantum Dots and Single Molecule Detection Enable High Sensitive Screening of Genetic Cancer Biomarkers

Jeff T.H. Wang
Associate Professor
Departments of Mechanical Engineering and Biomedical Engineering
Johns Hopkins University

Genomic analysis of biomarkers, including genetic markers such as point mutations and epigenetic markers such as DNA methylation, has become a central theme in modern disease diagnosis and prognosis. Recently there is an increasing interest in using confocal single-molecule spectroscopy (SMS) for genomic detection. The driving force not only comes from its ultrahigh sensitivity that allows detection of low-abundance nucleic acids without the need for amplification but also from its potential in achieving high-accuracy quantification of rare targets via single-molecule sorting. On the other hand, in recent years, semiconductor quantum dots (QDs) have attracted a great deal of attention for biomedical applications. The unique photophysical properties of semiconductor quantum dots (QDs) such as high quantum yield and photostability make them ideal for use as spectral labels and luminescent probes. QDs also make excellent donors to pair with organic dyes in the fluorescence resonance energy transfer (FRET) process due to the features of narrow emission spectra and small Stokes shift. This enables FRET with minimal direct acceptor excitation and donor-acceptor crosstalk, thereby permitting the design of FRET molecular sensors with extremely low intrinsic fluorescence backgrounds necessary for detecting biomolecular targets at low abundance. We have developed highly sensitive, quantitative and clinically relevant technologies for analysis of genomic markers based on the convergence of SMS, microfluidic manipulations, and quantum dot fluorescence resonance energy transfer technology (QD-FRET). Extraordinary performances of these new technologies have been exemplified by analysis of a variety of biomarkers including point mutations, DNA integrity and DNA methylation in clinical samples.

Nanotopographic surface for Efficient Capture of Circulating Tumor Cells

Weiqiang Chen
Graduate Student
Integrated Biosystems and Biomechanics Laboratory
Mechanical Engineering

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers.  Here we developed a novel CTC capture strategy that uniquely utilized thedifferential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cellsand thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.  Our cancer cell capture strategy using mechanosensitive properties of cancer cells could be applied to many different cancer cells derived from different human tissues with high capture yields.  In addition, our novel method can potentially provide a promising solution to study intratumor phenotypic heterogeneity at the single-cell level using patient CTCs for diagnostics and therapy.  Here we have further utilized microengineered tools for mechanophenotyping of cancer cells and cancer stem cells.  We have studied the cell deformability, adhesion strength, motility and contractility of different types of cancer cells and cancer stem cells.  Using mechanosensitive properties of cancer cells to achieve their efficient capture and mechanophenotyping could potentially lead to better informative analysis of CTCs for accurate diagnosis and prognosis of cancer and for fundamental understanding of cancer metastasis.

Maskless optofluidic synthesis of precision polymer microparticles for biointerfaces

C. Ryan Oliver
Graduate Student
Mechanosynthesis Group
Mechanical Engineering

Interfaces between biology and technology require new tools that leverage engineered shape and function to be compatible.  High precision nano/microparticle synthesis via optofluidics is a platform to build such tools.  This presentation will discuss work towards microparticle synthesis having arbitrary shape and chemistry using projection lithography coupled with a microfluidic device.  A re-configurable optical system using an integrated DMD chip to generates particle shapes on the fly (maskless) utilizes visible and UV light to polymerize PEG-DA within the microfluidic chip.  Several derivative techniques that utilize the re-configurability of the system will be presented for patterning onto surfaces.  Microparticles synthesized by optofluidics opens opportunities for study of non-spherical micro-particles for use in biointerfaces.

Multi-level microfluidic structures for cell trapping fabricated in a single exposure

Joe Labuz
Graduate Student
Takayama Lab
Biomedical Engineering

Backside Diffuse Light Lithography (BDLL) has been used to produce bell-shaped microfluidic channels that facilitate efficient valving in braille pumping systems. Recent work has leveraged BDLL techniques to generate small orrifices for droplet formation in a single exposure. This work builds on that concept, realizing two-layer structures fabricated in a single exposure using a variety of mask designs and demonstrates their utility in cell sorting applications. Furthermore, this method is possible without the use of a contact aligner or even collumated UV source, bringing BDLL-fabricated microfluidics within the grasp of many biology and applied science labs that would otherwise be unable to take advantage of photolithography-based microfluidics.

Bead Assembly Magnetorotation as a Biomarker Detection System

Ariel Hecht
Graduate Student
Kopelman Lab

As one of the many research groups working to design low-cost, portable, simple and inexpensive biomarker detection systems, we developed Bead Assembly Magnetorotation (BAM) as a tool to address this need. BAM is a unique detection method based on using the target biomarker to control the formation of magnetic beads into a two-dimensional assembly with fractal-like characteristics. The fractal dimension of the assembly depends on the concentration of the target biomarker, and the rotational period of the assembly in a rotating magnetic field depends on its fractal dimension. The advantages of BAM are the use of robust and inexpensive magnetic beads, a low power (5 mT) and low frequency (50 Hz) magnetic field, and a self-contained, low-power (20 mW) portable laser-and-photodiode detection platform. BAM performed very well in buffer, achieving a very low limit of detection (80 fM), fast analysis time (40 minutes), and low sample volume (100 μL). However, like many similar systems, significant challenges with nonspecific binding were encountered upon making the transition to serum. This presentation will discuss the accomplishments achieved in the development of BAM, as well as the challenges it faces in its attempt to move forward as a clinically useful system.

Developing Integrated Optofluidic Platforms for Cellular Immunophenotyping

Nien-Tsu (Joe) Huang
Graduate Student
Mechanical Engineering

Rapid, quantitative detection of cell-secreted biomarker proteins with a low sample volume holds great promise to advance cellular immunophenotyping techniques for personalized diagnosis and treatment of infectious diseases. Here we achieved such an assay with the THP-1 human moncytic cell line using a highly integrated microfluidic platform incorporating a no-wash bead-based chemiluminescence immunodetection scheme. Our microfluidic device allowed us to stimulate cells with lipopolysaccharide (LPS), which is an endotoxin causing septic shock due to severely pronounced immune response of the human body, under a well-controlled on-chip environment. Our study achieved high-sensitivity cellular immunophenotyping with 20-fold fewer cells than current cell-stimulation assay. Our strategy of monitoring immune cell functions in situ using a microfluidic platform could impact future medical treatments of acute infectious diseases and immune disorders by enabling a rapid, sample-efficient cellular immunophenotyping analysis.

Dual-pore glass chips for single-channel recording

Brandon R. Bruhn
Graduate Student
Biomembrane Lab
Biomedical Engineering

Despite the widespread use of high-throughput planar patch-clamp instruments, the conventional pipette-based technique remains the method of choice for recording single-channel activity. Generally, planar platforms are not well suited for single-channel studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms typically use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, thereby reducing the seal resistance. Here, femtosecond laser ablation is used to fabricate dual-pore glass chips for use in low-noise, single-channel recordings that circumvent this problem. One pore positions a cell by suction while another nearby pore, the recording pore, avoids contamination by maintaining positive pressure until a cell is positioned and then establishes a seal. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (>10 GΩ), maintained gigaseals for prolonged durations (up to 6 hrs), and enabled single-channel recordings in cell-attached mode that are comparable to those obtained by conventional patch-clamp.

Microfluidic source-sink-migration model: CXCR7 orchestrates CXCL12-isoform gradient formation and chemotaxis of CXCR4+ breast cancer cells

Steve Cavnar
Graduate Student
Takayama Lab
Biomedical Engineering

The chemokine CXCL12 is involved in the progression and metastasis in 20+ cancer types. Most studies of CXCL12 focus on the α-isoform of the ligand. However, 6 human isoforms are expressed in time- and organ-specific manners throughout development and disease. We used a novel microfluidic source-sink-migration model of CXCL12-isoform secretion (source cells), scavenging via CXCR7 (sink cells), and signaling via CXCR4 (migrating cells) to identify isoform-specific roles in metastasis. We used time point and kinetic tracking of CXCR4+ cells to reveal that, opposite to standard transwells, CXCL12-γ induced more chemotaxis than α- and β-isoforms. CXCL12-γ drove higher recruitment of β-arrestin-2 via CXCR4, lower recruitment of β-arrestin-2 via CXCR7, and lower CXCR7-dependent scavenging relative to other isoforms. By using scavenging-incompetent CXCR7 and by diluting the fraction of CXCL12-secreting cells we found two regimes of CXCR7 relevance in chemotaxis: 1) high CXCL12 requires CXCR7-scavenging, and 2) low CXCL12 bypasses the need for CXCR7-dependent scavenging. Treatment with clinical antagonist AMD3100 was unable to fully inhibit chemotaxis towards high levels of CXCL12-α and was surprisingly ineffective at inhibiting even low levels of β- and γ-isoforms. We found transcript expression of all three isoforms in tumors and bone marrow of relevant mouse models. In human tissues, CXCL12-α and -β were expressed in all stages of breast cancer, whereas the γ-isoform was only expressed in advanced stage III and IV. We found that despite lower reported affinity for its receptor CXCR4, CXCL12-γ acted as a high-affinity ligand and may have a distinct role in cancer metastasis.

Multicompartmental Polymer Carriers for Interfacing with Biological Environments

Asish Misra
Graduate Student
Lahann Lab
Biomedical Engineering

There is great potential for polymer micro- and nano- carriers in biomedical applications such as tissue engineering or drug delivery. However, while such technologies are hypothesized in some case to increase efficacy and potency of small molecule drugs this goal has not been realized. There are many barriers to effective therapy caused by both physiological and pathophysiological processes. Therefore, multifunctional carriers capable of addressing multiple challenges are required for effective therapy. Electrohydrodynamic co-jetting is a technique that may allow for the manufacturing of such particles. Here we propose to develop several particle systems using the co-jetting technique to address the challenge of developing carriers that can cope with these barriers to effective therapy.

Engineering Approaches to Regulate Immunity

James Moon
Assistant Professor
Pharmaceutical Sciences and Biomedical Engineering
University of Michigan

The immune system is a complex network of cells and organs that can detect and eliminate foreign pathogens by eliciting local and systemic immune responses. If we can engineer strategies to harness the potential of our own immune system, these new therapeutic approaches will transform the field of biomedicine, ranging from vaccines against infectious diseases to immunotherapies for cancer and autoimmunity. In this talk, I will discuss how fundamental principles of engineering in drug delivery and materials science can be utilized to control and manipulate the immune system. Specifically, I will describe our research efforts directed towards 1) the development of biologically-inspired nanoparticles that can mimic the key features of microbes to activate T-cells and B-cells, and 2) application of these nanoparticle vaccines in vivo to generate potent cellular and humoral immune responses against pathogens, including malaria sporozoites and HIV. These new synthetic materials coupled with a powerful set of engineering tools offer unique opportunities to advance our understanding of the immune system and translate discoveries from basic immunology towards new diagnostics and immunotherapies.

Nanopores with Fluid Walls for the Characterization of Single Proteins and Peptides

Erick Yusko
Graduate Student
Mayer Lab
Biomedical Engineering

Nanopore-based, resistive-pulse sensing is one of the simplest single-molecule techniques, since it is label free and employs basic electronic recording equipment. The technique involves measuring the ionic current through an electrolyte-filled nanopore and, when an insulating particle passes through the pore, detecting a transient decrease in this current. Sensing single proteins with this technique, however, is limited by transit times of proteins through the nanopore that are too fast to be resolved, non-specific interactions between the nanopore walls and the proteins, and poor specificity for the proteins being detected. This presentation introduces the concept of nanopores with fluid walls and their applications in single-molecule sensing of proteins and Alzheimer's disease-relevant aggregates of amyloid-β peptides. Inspired by the lipid-coated nanostructures that are found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to nanopores such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision and an ability to monitor membrane-active enzymes such as phospholipase D. Using these bilayer-coated nanopores with lipids presenting a ligand, specific proteins binding to the ligand were captured, concentrated on the surface, and preferentially transported to the nanopore, thereby, conferring specificity to a nanopore. Additionally, this strategy enabled well-defined control of protein transit times through the nanopore based on the viscosity of the fluid lipid coating and the first combined determination of a protein's volume, shape, charge, and affinity for a ligand using a single molecule technique. The fluid, biomemtic surface of a bilayer-coated nanopore was also non-fouling and enabled direct characterization of amyloid-β aggregates. The presented technique and analysis of these aggregates fulfills a previously unmet need in the amyloid research field: a method capable of determining the true size distributions and concentrations of amyloid-β aggregates in aqueous solution. The experiments and results that will be presented demonstrate how the concept of a nanopore with fluid walls enables nanopore-based assays that were previously inaccessible. In particular, it demonstrates the benefits of the fluid-wall concept for single-molecule studies of native proteins. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single-molecule assays.