Drug resistance is a characteristic of tumor initiating cells that can give rise to metastatic disease. In this work we demonstrate the use of microbubble well arrays as a cell culture platform to enumerate and characterize drug resistant cells in a human derived tumorigenic squamous cell carcinoma cell line. The spherical architecture and compliant hydrophobic composition of the microbubble well favors single cell survival, clonal proliferation and formation of spheres that do not grow on standard tissue culture plastic and are resistant to cisplatin. Spheres form in isolation and in microbubble wells containing proliferating cells and to some degree they stain positive for common stem cell markers CD44 and CD133. Spheres are also observed in cellularized primary human tumors cultured in microbubble arrays. This proof-of-concept study illustrates the potential for microbubble array technology to enumerate cancer cells resistant to standard care drugs with the ability to test alternative drug combinations. This capability can be developed for designing patient specific treatment strategies. Recovery of drug-resistant cells will allow a more full characterization of their gene expression profile thereby expanding our fundamental knowledge and ability to develop new targets to fight metastatic disease.

Previous work has demonstrated size, surface charge and skin barrier dependent penetration of nanoparticles into the viable layers of mouse skin. The goal of this work was to characterize the tissue distribution and mechanism of transport of nanoparticles beyond skin, with and without Ultraviolet Radiation (UVR) induced skin barrier disruption. Atomic absorption spectroscopy (AAS), flow cytometry and confocal microscopy were used to examine the effect of UVR dose (180 and 360 mJ/cm2 UVB) on the skin penetration and systemic distribution of quantum dot (QD) nanoparticles topically applied at different time-points post UVR using a hairless C57BL/6 mouse model.Results indicate that QDs can penetrate mouse skin, regardless of UVR exposure, as evidenced by the increased cadmium in the local lymph nodes of all QD treated mice. The average % recovery for all treatment groups was 69.68% with ~66.84% of the applied dose recovered from the skin (both epicutaneous and intracutaneous). An average of 0.024% of the applied dose was recovered from the lymph nodes across various treatment groups. When QDs are applied 4 days post UV irradiation, at the peak of the skin barrier defect and LC migration to the local lymph node, there is an increased cellular presence of QD in the lymph node; however, AAS analysis of local lymph nodes display no difference in cadmium levels due to UVR treatment.Our data suggests that Langerhans cells (LCs) can engulf QDs in skin, but transport to the lymph node may occur by both cellular (dendritic and macrophage) and non-cellular mechanisms. It is interesting that these specific nanoparticles were retained in skin similarly regardless of UVR barrier disruption, but the observed skin immune cell interaction with nanoparticles suggest a potential for immunomodulation, which we are currently examining in a murine model of skin allergy.

The metastatic potential of cancer cells is an elusive property that is indicative of the later stages of cancer progression. The ability to distinguish between poorly and highly metastatic cells is invaluable for understanding the basic biology of cancer and to develop more treatments. In this paper, we exploit a A375 melanoma cell line series (A375P, A375MA1, A375MA2) that vary in metastatic potential, to demonstrate an in vitro screening assay using polydimethylsiloxane (PDMS) microbubble well arrays that can distinguish these cell lines by their growth characteristics in including morphology, migratory potential, and clonogenic potential. These cell lines cannot be distinguished by their growth characteristics when cultured on standard tissue culture plastic or planar PDMS. Results show that the more metastatic cell lines (A375MA1, A375MA2) have a higher proliferative potential and a distinctive radial spreading growth pattern out of the microbubble well. The A375MA2 cell line also has a higher tendency to form multicellular spheroids. The ability to successfully correlate the metastatic potential of cancer cells with their growth characteristics is essential first step toward developing a high-throughput screening assay to identify aggressive tumor cells in primary samples. The capability to culture and recover aggressive cells from microbubble wells will enable identification of candidate metastatic biomarkers which has immense clinical significance.

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.

The therapeutic potential of monoclonal antibodies (mAbs) makes them an ideal tool in both clinical and research applications due to their ability to recognize and bind specific epitopes with high affinity and selectivity. While mAbs offer significant therapeutic potential, their utility is overshadowed by the cost associated with their production, which often relies on the ability to identify minor antigen-specific cells out of a heterogeneous population. To address concerns with suboptimal methods for screening cells, we have developed a cell-sorting array composed of nanoliter spherical cell culture compartments termed microbubble (MB) wells. We demonstrate a proof-of-concept system for the detection of cell secreted factors from both immortalized cell lines and primary B cell samples. Exploiting the unique ability of the MB well architecture to accumulate cell secreted factors as well as affinity capture coatings, we demonstrate on-chip detection and recovery of antibody-secreting cells for sequencing of immunoglobin genes. Furthermore, rapid image capture and analysis capabilities were developed for the processing of large MB arrays, thus facilitating the ability to conduct high-throughput screening of heterogeneous cell samples faster and more efficiently than ever before. The proof-of-concept assays presented herein lay the groundwork for the progression of MB well arrays as an advanced on-chip cell sorting technology.

Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in2). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in2 with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.

Development of micro-well array systems for use in high-throughput screening of rare cells requires a detailed understanding of the factors that impact the specific capture of cells in wells and the distribution statistics of the number of cells deposited into wells. In this study we investigate the development of microbubble (MB) well array technology for sorting antigen-specific B-cells. Using Poisson statistics we delineate the important role that the fractional area of MB well opening and the cell seeding density have on determining cell seeding distribution in wells. The unique architecture of the MB well hinders captured cells from escaping the well and provides a unique microenvironmental niche that enables media changes as needed for extended cell culture. Using cell lines and primary B and T cells isolated from human peripheral blood we demonstrate the use of affinity capture agents coated in the MB wells to enrich for the selective capture of B cells. Important differences were noted in the efficacy of bovine serum albumin to block the nonspecific adsorption of primary cells relative to cell lines as well as the efficacy of the capture coatings using mixed primary B and T cells samples. These results emphasize the importance of using primary cells in technology development and suggest the need to utilize B cell capture agents that are insensitive to cell activation.

In this work, we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n = 50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was <9% both within-run and between-day at and above the cutoff limit of 300 ng mL−1. This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semiquantitative demonstration of the technology’s successful clinical use. These results motivate future development of label-free PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting.

Currently, there is need for laboratory-based high-throughput and reliable point-of-care drug screening methodologies. We demonstrate here a chip-based label-free porous silicon (PSi) photonic sensor for detecting opiates in urine. This technique provides a cost-effective alternative to conventional labeled drug screening immunoassays with potential for translation to multiplexed analysis. Important effects of surface chemistry and competitive binding assay protocol on the sensitivity of opiate detection are revealed. Capability to tune sensitivity and detection range over ∼3 orders of magnitude (18.0 nM to 10.8 μM) was achieved by varying the applied urine specimen volume (100−5 μL), which results in systematic shifts in the competitive binding response curve. A detection range (0.36−4.02 μM) of morphine in urine (15 μL) was designed to span the current positive cutoff value (1.05 μM morphine) in medical opiate urine screening. Desirable high cross-reactivity to oxycodone, in addition to other common opiates, morphine, morphine-3-glucuronide, 6-acetyl morphine, demonstrates an advantage over current commercial screening assays, while low interference with cocaine metabolite was maintained. This study uniquely displays PSi sensor technology as an inexpensive, rapid, and reliable drug screening technology. Furthermore, the versatile surface chemistry developed can be implemented on a range of solid-supported sensors to conduct competitive inhibition assays.

Ultraviolet radiation (UVR) has widespread effects on the biology and integrity of the skin barrier. Research on the mechanisms that drive these changes, as well as their effect on skin barrier function, has been ongoing since the 1980s. However, no studies have examined the impact of UVR on nanoparticle skin penetration. Nanoparticles (NP) are commonly used in sunscreens and other cosmetics, and since consumer use of sunscreen is often applied to sun damaged skin, the effect of UVR on NP skin penetration is a concern due to potential toxicity. In this study, we investigate NP skin penetration by employing an in vivo semiconductor quantum dot nanoparticle (QD) model system. This model system improves NP imaging capabilities and provides additional primary interest due to widespread and expanding use of QD in research applications and manufacturing. In our experiments, carboxylated QD were applied to the skin of SKH-1 mice in a glycerol vehicle with and without UVR exposure. The skin collection and penetration patterns were evaluated 8 and 24 h after QD application using tissue histology, confocal microscopy, and transmission electron microscopy (TEM) with EDAX analysis. Low levels of penetration were seen in both the non-UVR exposed mice and the UVR exposed mice. Qualitatively higher levels of penetration were observable in the UVR exposed mice. These results are the first for in vivo QD skin penetration, and provide important insight into the ability of QD to penetrate intact and UVR compromised skin barrier. Our findings raise concern that NP of similar size and surface chemistry, such as metal oxide NP found in sunscreens, may also penetrate UV damaged skin.

We present a novel method to create cavities in PDMS that is simple and exhibits wide process latitude allowing control over the radius of curvature to form shallow concave pits or deep spherical cavities.

The future of rapid point-of-care diagnostics depends on the development of cheap, noncomplex, and easily integrated systems to analyze biological samples directly from the patient (e.g. blood, urine, and saliva). A key concern in diagnostic biosensing is signal differentiation between non-specifically bound material and the specific capture of target molecules. This is a particular challenge for optical detection devices in analyzing complex biological samples. Here we demonstrate a porous silicon (PSi) label-free optical biosensor that has intrinsic size-exclusion filtering capabilities which enhances signal differentiation. We present the first demonstration of highly repeatable, specific detection of immunoglobulin G (IgG) in serum and whole blood samples over a typical physiological range using the PSi material as both a biosensor substrate and filter.

What are nanoparticles and why are they important in dermatology? These questions are addressed by highlighting recent developments in the nanotechnology field that have increased the potential for intentional and unintentional nanoparticle skin exposure. The role of environmental factors in the interaction of nanoparticles with skin and the potential mechanisms by which nanoparticles may influence skin response to environmental factors are discussed. Trends emerging from recent literature suggest that the positive benefit of engineered nanoparticles for use in cosmetics and as tools for understanding skin biology and curing skin disease outweigh potential toxicity concerns. Discoveries reported in this journal are highlighted. This review begins with a general introduction to the field of nanotechnology and nanomedicine. This is followed by a discussion of the current state of understanding of nanoparticle skin penetration and their use in three therapeutic applications. Challenges that must be overcome to derive clinical benefit from the application of nanotechnology to skin are discussed last, providing perspective on the significant opportunity that exists for future studies in investigative dermatology.

We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.