Daniel T. Chiu

Fabrication of microfluidic systems in poly(dimethylsiloxane).

Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft‐lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics‐derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine

In recent years, semiconducting polymer nanoparticles have attracted considerable attention because of their outstanding characteristics as fluorescent probes. These nanoparticles, which primarily consist of π-conjugated polymers and are called polymer dots (Pdots) when they exhibit small particle size and high brightness, have demonstrated utility in a wide range of applications such as fluorescence imaging and biosensing. In this review, we summarize recent findings of the photophysical properties of Pdots which speak to the merits of these entities as fluorescent labels. This review also highlights the surface functionalization and biomolecular conjugation of Pdots, and their applications in cellular labeling, in vivo imaging, single-particle tracking, biosensing, and drug delivery. We discuss the relationship between the physical properties and performance, and evaluate the merits and limitations of the Pdot probes for certain imaging tasks and fluorescence assays. We also tackle the current challenges of Pdots and share our perspective on the future directions of the field.

Bioconjugation of ultrabright semiconducting polymer dots for specific cellular targeting.

Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, nonblinking, and nontoxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is co-condensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable nonspecific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-μm widths were readily traversed by the 8 μm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-μm channels; some that passed through the 4-μm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-μm channels, trophozoites mimicked “pitting,” a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-μm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-μm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

Disposable microfluidic devices: fabrication, function, and application.

This review article describes recent developments in microfluidics, with special emphasis on disposable plastic devices. Included is an overview of the common methods used in the fabrication of polymer microfluidic systems, including replica and injection molding, embossing, and laser ablation. Also described are the different methods by which on-chip operations--such as the pumping and valving of fluid flow, the mixing of different reagents, and the separation and detection of different chemical species--have been implemented in a microfluidic format. Finally, a few select biotechnological applications of microfluidics are presented to illustrate both the utility of this technology and its potential for development in the future.

Ratiometric Temperature Sensing with Semiconducting Polymer Dots

This communication describes ultrabright single-nanoparticle ratiometric temperature sensors based on semiconducting polymer dots (Pdots). We attached the temperature sensitive dye-Rhodamine B (RhB), whose emission intensity decreases with increasing temperature-within the matrix of Pdots. The as-prepared Pdot-RhB nanoparticle showed excellent temperature sensitivity and high brightness because it took advantage of the light harvesting and amplified energy transfer capability of Pdots. More importantly, the Pdot-RhB nanoparticle showed ratiometric temperature sensing under a single wavelength excitation and has a linear temperature sensing range that matches well with the physiologically relevant temperatures. We employed Pdot-RhB for measuring intracellular temperatures in a live-cell imaging mode. The exceptional brightness of Pdot-RhB allows this nanoscale temperature sensor to be used also as a fluorescent probe for cellular imaging.

Design of Highly Emissive Polymer Dot Bioconjugates for In Vivo Tumor Targeting

Nanoparticle-based diagnostic and therapeutic agents have attracted considerable interest because of their potential for clinical oncology and other biomedical research.[1] Versatile nanostructures have been demonstrated for in vivo applications, such as lipid and polymeric nanocapsules for drug delivery,[2] iron oxide nanoparticles for magnetic resonance imaging,[3] gold nanoparticles for X-ray computed tomography,[4] and quantum dots (Qdots) for fluorescence imaging.[5] Qdots represent one of the exciting nanotechnologies translated to biology in the past decade. The size-tunable luminescence makes them appealing as multicolor fluorophores for biological labelling, imaging, and sensing.[6,7] For in vivo applications, however, the intrinsic toxicity of Qdots is of critical concern,[8] which may impede their final clinical translation. Therefore, the design of bright probes with biologically benign materials is highly desirable for many in vivo clinical applications.

In Vivo Dynamic Monitoring of Small Molecules with Implantable Polymer-Dot Transducer

Small molecules participate extensively in various life processes. However, specific and sensitive detection of small molecules in a living system is highly challenging. Here, we describe in vivo real-time dynamic monitoring of small molecules by a luminescent polymer-dot oxygen transducer. The optical transducer combined with an oxygen-consuming enzyme can sensitively detect small-molecule substrates as the enzyme-catalyzed reaction depletes its internal oxygen reservoir in the presence of small molecules. We exemplify this detection strategy by using glucose-oxidase-functionalized polymer dots, yielding high selectivity, large dynamic range, and reversible glucose detection in cell and tissue environments. The transducer-enzyme assembly after subcutaneous implantation provides a strong luminescence signal that is transdermally detectable and continuously responsive to blood glucose fluctuations for up to 30 days. In view of a large library of oxygen-consuming enzymes, this strategy is promising for in vivo detection and quantitative determination of a variety of small molecules.

Microfluidics Section: Design and Fabrication of Integrated Passive Valves and Pumps for Flexible Polymer 3-Dimensional Microfluidic Systems

This paper describes the fabrication of flexible, polymeric 3-dimensional microfluidic systems with integrated check valves (flap and diaphragm valves) and a pump by stacking patterned poly(dimethylsiloxane) (PDMS) layers containing microchannels and vias. We describe this procedure for fabricating, manipulating, and bonding of PDMS membranes and bas-relief plates into multilayer microfluidic devices. The fabrication and demonstration of integrated check valves and a pump in a prototype polymer 3-dimensional microfluidic system is a step toward practical realization of all-polymer, flexible, low-cost, disposable microfluidic devices for biochemical applications.

Near-Infrared Fluorescent Dye-Doped Semiconducting Polymer Dots

Near-infrared (NIR) fluorescence sensing is desirable for in vivo biological measurements, but the method is currently limited by the availability of NIR fluorescent markers as well as by their poor performance, such as self-aggregation and dim fluorescence, in a physiological environment. To address this issue, this paper describes a NIR fluorescent polymer dot (Pdot) that emits at 777 nm. This Pdot was comparable in size to a water-soluble NIR quantum dot that emits at 800 nm (ITK Qdot800) but was about four times brighter and with a narrower emission peak. We formed the NIR Pdot by doping the NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), into the matrix of poly (9,9-dioctylfluorene-co-benzothiadiazole) (PFBT) as the Pdot formed using a nanoscale precipitation technique. Free molecules of NIR775 aggregate in aqueous solution, but encapsulating them into the hydrophobic Pdot matrix effectively introduced them into aqueous solution for use in biological studies. Most importantly, the brightness of NIR775 was dramatically enhanced because of the excellent light-harvesting ability of PFBT and the very efficient energy transfer from PFBT to NIR775. We anticipate this bright NIR Pdot will be useful in biological measurements and cellular imaging where strong NIR emission is beneficial.