Barbara S. Smith

Folding analytical devices for electrochemical ELISA in hydrophobic R(H) paper.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.

Hemocompatibility of titania nanotube arrays

Hemocompatibility is a key consideration for the long-term success of blood contacting biomaterials; hence, there is a critical need to understand the physiological response elicited from blood/nano-biomaterial interactions. In this study, we have investigated the adsorption of key blood serum proteins, in vitro adhesion and activation of platelets, and clotting kinetics of whole blood on titania nanotube arrays. Previous studies have demonstrated improved mesenchymal stem cell functionality, osteoblast phenotypic behavior, localized drug delivery, and the production of endothelial cell ECM on titania nanotube arrays. Furthermore, these titania nanotube arrays have elicited minimal levels of monocyte activation and cytokine secretion, thus exhibiting a very low degree of immunogenicity. Titania nanotube arrays were fabricated using anodization technique and the surface morphology was examined through scanning electron microscopy (SEM). The crystalline phases were identified using glancing angled X-ray diffraction (GAXRD). Nanoindentation and scratch tests were used to characterize the mechanical properties of titania nanotube arrays. The adsorption of key blood proteins (albumin, fibrinogen, and immunoglobulin-g) was evaluated using a micro-BCA assay and X-ray photoelectron spectroscopy (XPS). The adhesion and activation of platelets was investigated using live-cell staining, MTT assay, and SEM. Whole blood clotting kinetics was evaluated by measuring the free hemoglobin concentration, and SEM was used to visualize the clot formation. Our results indicate increased blood serum protein adsorption, platelet adhesion and activation, and whole blood clotting kinetics on titania nanotube arrays.

Dermal fibroblast and epidermal keratinocyte functionality on titania nanotube arrays

Transcutaneous implants that penetrate through the depth of the skin are used in numerous clinical applications, including prosthetics and dental implants. Favorable interactions between the implant surface and the respective skin layers are critical for the long-term success of transcutaneous implantable devices, hence, it is essential to understand the physiologic response elicited by skin-biomaterial interactions. Recent studies have shown that material surfaces that provide topographic cues at the nanoscale level may provide one possible solution to enhanced biomaterial integration, thus preventing biomaterial rejection. In this study titania nanotube arrays were fabricated using a simple anodization technique as potential interfaces for transcutaneous implantable devices. The in vitro functionality of human dermal fibroblasts and epidermal keratinocytes were evaluated on these nanotube arrays (diameter 70-90 nm, length 1-1.5 μm). Cellular functionality in terms of adhesion, proliferation, orientation, viability, cytoskeletal organization, differentiation and morphology were investigated for up to 4 days in culture using fluorescence microscope imaging, a cell viability assay, indirect immunofluorescence and scanning electron microscopy. The results reported in this study indicate increased dermal fibroblast and decreased epidermal keratinocyte adhesion, proliferation and differentiation on titania nanotube arrays.

Reduced in vitro immune response on titania nanotube arrays compared to titanium surface

Material surfaces that provide biomimetic cues, such as nanoscale architectures, have been shown to alter cell/biomaterial interactions. Recent studies have identified titania nanotube arrays as strong candidates for use in interfaces on implantable devices due to their ability to elicit improved cellular functionality. However, limited information exists regarding the immune response of nanotube arrays. Thus, in this study, we have investigated the short- and long-term immune cell reaction of titania nanotube arrays. Whole blood lysate (containing leukocytes, thrombocytes and trace amounts of erythrocytes), isolated from human blood, were cultured on titania nanotube arrays and biomedical grade titanium (as a control) for 2 hours and 2 and 7 days. In order to determine the in vitro immune response on titania nanotube arrays, immune cell functionality was evaluated by cellular viability, adhesion, proliferation, morphology, cytokine/chemokine expression, with and without lipopolysaccharide (LPS), and nitric oxide release. The results presented in this study indicate a decrease in short- and long-term monocyte, macrophage and neutrophil functionality on titania nanotube arrays as compared to the control substrate. This work shows a reduced stimulation of the immune response on titania nanotube arrays, identifying this specific nanoarchitecture as a potentially optimal interface for implantable biomedical devices.

Hemocompatibility of polymeric nanostructured surfaces

Tissue integration is an important property when inducing transplant tolerance, however, the hemocompatibility of the biomaterial surface also plays an important role in the ultimate success of the implant. Therefore, in order to induce transplant tolerance, it is critical to understand the interaction of blood components with the material surfaces. In this study, we have investigated the adsorption of key blood serum proteins, in vitro adhesion and activation of platelets and clotting kinetics of whole blood on flat polycaprolactone (PCL) surfaces, nanowire (NW) surfaces and nanofiber (NF) surfaces. Previous studies have shown that polymeric nanostructured surfaces improve cell adhesion, proliferation and viability; however it is unclear how these polymeric nanostructured surfaces interact with the blood and its components. Protein adsorption results indicate that while there were no significant differences in total albumin (ALB) adsorption on PCL, NW and NF surfaces, NW surfaces had higher total fibrinogen (FIB) and immunoglobulin-G (IgG) adsorption compared to NF and PCL surfaces. In contrast, NF surfaces had higher surface FIB and IgG adsorption compared to PCL and NW surfaces. Platelet adhesion and viability studies show more adhesion and clustering of platelets on the NF surfaces as compared to PCL and NW surfaces. Platelet activation studies reveal that NW surfaces have the highest percentage of unactivated platelets, whereas NF surfaces have the highest percentage of fully activated platelets. Whole blood clotting results indicate that NW surfaces maintain an increased amount of free hemoglobin during the clotting process compared to PCL and NF surface, indicating less clotting and slower rate of clotting on their surfaces.

Correlation of mechanical properties within the equine third metacarpal with trabecular bending and multi-density micro-computed tomography data.

Computed tomography (CT) data can be employed with respect to determining mechanical properties and has been used to predict parameters such as elastic modulus, yield strength, and ultimate strength of intact bone. Micro-computed tomography (muCT) possesses the resolution capable of detecting apparent bone density in extremely local regions and can characterize the trabecular structure. It has been asserted that this micro-structure is susceptible to micro-buckling and bending, which has a controversial role in predicting the global mechanical properties of bone. The current study measured the mechanical properties of relatively high apparent density bone from the equine distal third metacarpal. The mechanical properties were correlated with trabecular morphology parameters and apparent densities of localized regions obtained with muCT. These data were used to test two hypotheses: (1) accounting for trabecular bending using trabecular morphology parameters would provide better global mechanical property predictions than using only apparent density, and (2) regions of low apparent density dominate the overall mechanical behavior and provide greater correlation to the measured mechanical properties than regions of high apparent density. The data indicated that accounting for trabecular bending with morphological parameters resulted in stronger correlations to mechanical properties than correlations that relied only on apparent density (r2= 0.91 versus r2= 0.81). Low apparent density regions were more strongly correlated with mechanical properties than high apparent density regions (r2= 0.85 versus r2= 0.77), demonstrating the importance of selecting appropriate regions when attempting to predict mechanical properties from CT data.

Cellular Functionality on Nanotubes of Ti-30Ta Alloy

Recent studies have identified strong correlations between anodized metals and the production of highly biomimetic nanoscale topographies. These surfaces provide an interface of enhanced biocompatibility that exhibits a high degree of oxidation and surface energy. In this study, Human dermal fibroblasts (HDF, neonatal) were utilized to evaluate the biocompatibility of Ti-30Ta nanotubes after 1 day of culture. The anodization process was performed in an electrolyte solution containing HF (48%) and H2SO4 (98%) in the volumetric ratios 1:9 with the addition of 5% dimethyl sulfoxide (DMSO) at 35V for 40 min. Cellular analysis identified improved fibroblast functionality on the nanotube surface, showing increased elongation, and extracellular matrix production on the Ti-30Ta nanotubes. The results presented identify improved cellular interaction on Ti-30Ta nanotubes as compared to the control substrates. Thus, the formation of the nanotube on Ti30Ta alloy may have potential application as interface for implantable devices.

Fluorescence Immunoassay for the Detection of Latent Tuberculosis Antigens with Single Molecule Sensitivity

The successful identification and detection at the single molecule level of Antigen 85b, an antigen released by tuberculosis, was accomplished using a fluorescence-based immunoassay. This work enables a method for the diagnosis of latent tuberculosis.