Samuel P. Forry

Dynamics and mechanisms of quantum dot nanoparticle cellular uptake

BackgroundThe rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.ResultsWe explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC), we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM) images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h.ConclusionsThe findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

A noninvasive thin film sensor for monitoring oxygen tension during in vitro cell culture.

Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viability, and proliferation. However, precise measurement of dissolved oxygen in real time remains difficult. We report a new noninvasive sensor that can accurately measure oxygen concentration during cell culture while being compatible with live-cell imaging techniques such as fluorescence and phase contrast microscopy. The sensor is prepared by integrating the porphyrin dye, Pt(II) meso-tetrakis(pentafluorophenyl)porphine (PtTFPP) into polydimethylsiloxane (PDMS) thin films. Response of the sensor in the presence of oxygen can be characterized by the linear Stern-Volmer relationship with high sensitivity (K(SV) = 584 +/- 71 atm(-1)). A multilayer sensor design, created by sandwiching the PtTFPP-PDMS with a layer of Teflon AF followed by a second PDMS layer, effectively mitigates against dye cytotoxicity while providing a substrate for cell attachment. Using this sensor, changes in oxygen tension could be monitored in real-time as attached cells proliferated. The oxygen tension was found to decrease due to oxygen consumption by the cells, and the data could be analyzed using Ficks law to obtain the per-cell oxygen consumption rate. This sensor is likely to enable new studies on the effects of dissolved oxygen on cellular behavior.

Regulating oxygen levels in a microfluidic device.

Microfluidic devices made from poly(dimethylsiloxane) (PDMS) are gas permeable and have been used to provide accurate on-chip oxygen regulation. However, pervaporation in PDMS devices can rapidly lead to dramatic changes in solution osmotic pressure. In the present study, we demonstrate a new method for on-chip oxygen control using pre-equilibrated aqueous solutions in gas-control channels to regulate the oxygen content in stagnant microfluidic test chambers. An off-chip gas exchanger is used to equilibrate each control solution prior to entering the chip. Using this strategy, problems due to pervaporation are considerably reduced. An integrated PDMS-based oxygen sensor allows accurate real-time measurements of the oxygen within the microfluidic chamber. The measurements were found to be consistent with predictions from finite-element modeling.

Microfluidic synthesis of monodisperse PDMS microbeads as discrete oxygen sensors

We describe the creation of monodisperse microbeads of polydimethylsiloxane (PDMS) via a microfluidic approach. Using a flow-focusing configuration, a PDMS precursor solution is dispersed into microdroplets within an aqueous continuous phase. These droplets are collected and thermally cured off-chip into solid microbeads. Our microfluidic technique allows for direct integration of payloads into the PDMS microbeads. Specifically, we integrate an oxygen-sensitive porphyrin dye into the beads and show that the resulting structures can function as non-invasive and real-time oxygen microsensors utilizing a simple optical readout at the single-particle level.

On-chip CO2 control for microfluidic cell culture

Carbon dioxide partial pressure (P(CO(2))) was controlled on-chip by flowing pre-equilibrated aqueous solutions through control channels across the device. Elevated P(CO(2)) (e.g. 0.05 atm) was modulated in neighboring stagnant channels via equilibration through the highly gas permeable substrate, poly(dimethylsiloxane) (PDMS). Stable gradients in P(CO(2)) were demonstrated with a pair of control lines in a source-sink configuration. P(CO(2)) equilibration was found to be sufficiently rapid (minutes) and stable (days) to enable long-term microfluidic culture of mammalian cells. The aqueous solutions flowing through the device also mitigated pervaporative losses at sustained elevated temperatures (e.g. 37 C), as compared to flowing humidified gas through the control lines to control P(CO(2)). Since pervaporation (and the associated increase in osmolality) was minimized, stopped-flow cell culture became possible, wherein cell secretions can accumulate within the confined environment of the microfluidic culture system. This strategy was utilized to demonstrate long-term (> 7 days) microfluidic culture of mouse fibroblasts under stopped-flow conditions without requiring the microfluidic system to be placed inside a cell culture incubator.

Dielectrophoretic capture of mammalian cells using transparent indium tin oxide electrodes in microfluidic systems.

Transparent indium tin oxide microelectrodes were fabricated and used to immobilize suspended NIH 3T3 fibroblast cells by positive dielectrophoresis. The indium tin oxide electrodes facilitated microscopic observation of immobilized cells compared with opaque metallized electrodes. Dielectrophoresis was used to capture arrays of individual cells and form small cell clusters within a microfluidic network. The extent of cellular immobilization (no‐cell, single‐cell, or multiple‐cell capture) was correlated with the applied voltage and inversely with the flow velocity. Specific conditions yielding predominantly single‐cell capture were identified. The viability of immobilized cells was confirmed using fluorescence microscopy.

Capturing rare cells from blood using a packed bed of custom-synthesized chitosan microparticles

We describe batch generation of uniform multifunctional chitosan microparticles for isolation of rare cells, such as circulating tumor cells (CTCs), from a sample of whole blood. The chitosan microparticles were produced in large numbers using a simple and inexpensive microtubing arrangement. The particles were functionalized through encapsulation of carbon black, to control autofluorescence, and surface attachment of streptavidin, to enable interactions with biotinylated antibodies. These large custom modified microparticles (≈164 μm diameter) were then packed into a microfluidic channel to demonstrate their utility in rare cell capture. Blood spiked with breast cancer (MCF-7) cells was first treated with a biotinylated antibody (anti-EpCAM, which is selective for cancer cells like MCF-7) and then pumped through the device. In the process, the cancer cells were selectively bound to the microparticles through non-covalent streptavidin-biotin interactions. The number density of captured cells was determined by fluorescence microscopy at physiologically relevant levels. Selective capture of the MCF-7 cells was characterized, and compared favorably with previous approaches. The overall approach using custom synthesized microparticles is versatile, and can allow researchers more flexibility for rare cell capture through simpler and cheaper methods than are currently employed.

Use of DigiSim to Model Cyclic Voltammetric and Photonic Responses in Electrogenerated Chemiluminescent Systems

We demonstrate the use of DigiSim, a commercially available computer code, to model both the cyclic voltammetric and photonic responses of electrogenerated chemiluminescent systems. With this approach we exploit the capability of DigiSim to simulate complex electrochemical behavior involving myriad coupled heterogeneous electron transfers and homogeneous reactions. The present work focuses on the simulation of a relatively simple mechanism for electrogenerating chemiluminescence. Use of multiple cycles and introduction of more complicated mechanisms, quasi-reversible electron transfer, uncompensated resistance, and capacitance entail straightforward modifications of the basic approach.

Theoretical analysis of a magnetophoresis-diffusion T-sensor immunoassay

We present the analytical investigation of a microfluidic homogeneous competitive immunoassay that incorporates antibody-conjugated superparamagnetic nanoparticles and magnetophoretic transport to enhance the limits of detection and dynamic range. The analytical model considers the advective, diffusive, and magnetophoretic transport of the antibody-coated nanoparticles relative to the labeled and sample antigens of interest in a T-sensor configuration. The magnetophoresis-diffusion immunoassay identified clear improvements to the assay response and reductions to the limit of detection for increased magnetophoretic velocities and larger nanoparticles. The externally applied magnetophoretic transport enriched the antibody-antigen accumulation region, while larger nanoparticles led to decreased diffusive peak broadening. The integration of nanoparticles to the diffusion immunoassay (NP-DIA) demonstrated an approximately 3-fold improvement to the limit of detection of the basic antibody/antigen system, while the integration of superparamagnetic nanoparticles and magnetophoretic transport (MIA) established an order of magnitude improvement in sensitivity as well as means to greatly reduce response time. The implementation of an external magnetic force enabled the detectable antigen size spectrum to extend from small molecules i.e., 10s Da to 100s Da, up to large proteins and macromolecules, i.e., 50 kDa to 150 kDa, for a single class of binding species, i.e., superparamagnetic nanoparticle. This investigation provides guidelines for the design and development of a magnetophoresis-diffusion T-sensor immunoassay, and clearly identifies the regimes for optimal operation.

Measurement and validation of cell-based assays with microfluidics at the National Institute of Standards and Technology.

The National Institute of Standards and Technology (NIST) is the National Metrology Institute for the USA. Our mission is to advance measurement science, standards and technology in ways that enhance economic security and improve quality of life in the USA. Due to the increased need for technologies that advance biological research and the many new and exciting innovations in microfluidics, our projects are aimed at engineering well-controlled microenvironments for quantitative measurements of cell behavior in microfluidic systems. Cell-based microfluidics at NIST is a highly multidisciplinary activity and is greatly influenced by NIST programs in biochemical sciences, materials science, engineering and information technology. Although there are many microfluidic-related activities ongoing at NIST, we will focus on projects related to cell-based measurements in this article.