Dong-Ku Kang

Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4u2009h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.

A polyvalent aptamer system for targeted drug delivery

Poor efficacy and off-target systemic toxicity are major problems associated with current chemotherapeutic approaches to treat cancer. We developed a new form of polyvalent therapeutics that is composed of multiple aptamer units synthesized by rolling circle amplification and physically intercalated chemotherapy agents (termed as Poly-Aptamer-Drug). Using a leukemia cell-binding aptamer and doxorubicin as a model system, we have successfully constructed Poly-Aptamer-Drug systems and demonstrated that the Poly-Aptamer-Drug is significantly more effective than its monovalent counterpart in targeting and killing leukemia cells due to enhanced binding affinity (≈ 40 fold greater) and cell internalization via multivalent effects. We anticipate that our Poly-Aptamer-Drug approach will yield new classes of tunable therapeutics that can be utilized to effectively target and treat cancers while minimizing the side effects of chemotherapy.

Novel Molecular and Nanosensors for In Vivo Sensing

In vivo sensors are an emerging field with the potential to revolutionize our understanding of basic biology and our treatment of disease. In this review, we highlight recent advances in the fields of in vivo electrochemical, optical, and magnetic resonance biosensors with a focus on recent developments that have been validated in rodent models or human subjects. In addition, we discuss major challenges in the development and translation of in vivo biosensors and present potential solutions to these problems. The field of nanotechnology, in particular, has recently been instrumental in driving the field of in vivo sensors forward. We conclude with a discussion of emerging paradigms and techniques for the development of future biosensors.

From Blood to the Brain: Can Systemically Transplanted Mesenchymal Stem Cells Cross the Blood-Brain Barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors.

DNA-Scaffolded Multivalent Ligands to Modulate Cell Function

We report a simple, versatile, multivalent ligand system that is capable of specifically and efficiently modulating cell‐surface receptor clustering and function. The multivalent ligand is made of a polymeric DNA scaffold decorated with biorecognition ligands (i.e., antibodies) to interrogate and modulate cell receptor signaling and function. Using CD20 clustering‐mediated apoptosis in B‐cell cancer cells as a model system, we demonstrated that our multivalent ligand is significantly more effective at inducing apoptosis of target cancer cells than its monovalent counterpart. This multivalent DNA material approach represents a new chemical biology tool to interrogate cell receptor signaling and functions and to potentially manipulate such functions for the development of therapeutics.

Cell-surface sensors: lighting the cellular environment

Cell-surface sensors are powerful tools to elucidate cell functions including cell signaling, metabolism, and cell-to-cell communication. These sensors not only facilitate our understanding in basic biology but also advance the development of effective therapeutics and diagnostics. While genetically encoded fluorescent protein/peptide sensors have been most popular, emerging cell surface sensor systems including polymer-, nanoparticle-, and nucleic acid aptamer-based sensors have largely expanded our toolkits to interrogate complex cellular signaling and micro- or nano-environments. In particular, cell-surface sensors that interrogate in vivo cellular microenvironments represent an emerging trend in the development of next generation tools which biologists may routinely apply to elucidate cell biology in vivo and to develop new therapeutics and diagnostics. This review focuses on the most recent development in areas of cell-surface sensors. We will first discuss some recently reported genetically encoded sensors that were used for monitoring cellular metabolites, proteins, and neurotransmitters. We will then focus on the emerging cell surface sensor systems with emphasis on the use of DNA aptamer sensors for probing cell signaling and cell-to-cell communication.

Floating Droplet Array: An Ultrahigh-Throughput Device for Droplet Trapping, Real-time Analysis and Recovery.

We describe the design, fabrication and use of a dual-layered microfluidic device for ultrahigh-throughput droplet trapping, analysis, and recovery using droplet buoyancy. To demonstrate the utility of this device for digital quantification of analytes, we quantify the number of droplets, which contain a β-galactosidase-conjugated bead among more than 100,000 immobilized droplets. In addition, we demonstrate that this device can be used for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction of β-galactosidase and its fluorogenic substrate FDG, between droplets.

Probe and Control of Cell–Cell Interactions Using Bioengineered Tools

Understanding how cells interact and communicate with each other in multicellular organisms is essential to understanding diverse life and biological processes. However, the complicated nature of cells, their various in vivo microenvironments, and the multitude of changing environmental and biological signals that confront cells make it challenging to probe and control cell–cell interaction and communication using conventional macro-scale and population-averaged tools. With the rapid advances in bioengineering, we now have ever finer tools to peek increasingly closely at cellular processes, including the interaction and communication of cells with each other. In this chapter, we discuss established as well as emerging micro-, nano-, and molecular-scale genetic, chemical, and engineering tools designed to address remaining challenges in cell–cell interactions and communication. Microengineering tools, such as microfluidic chips, microwells, and microdroplets, make possible the manipulation of the spatial and temporal distribution of individual cells or defined homotypic and heterotypic cell clusters, thus simplifying the complicated in vivo microenvironments into well-controlled in vitro platforms. Molecular tools based on genetic or chemical approaches, frequently utilizing fluorescent or luminescent reporter signals, are being used at increased resolution to interrogate target cells. Moreover, with advances in complementary imaging systems, such as two-photon imaging, it is now possible to probe cell–cell interactions in their in vivo microenvironments. The knowledge derived from these studies is expected to not only increase the information content on various biological processes but also reveal novel paradigms of cellular and physiological processes, thereby also helping in the design of novel strategies to diagnose and treat diseases.