This PDF file contains the front matter associated with SPIE Proceedings Volume 9930, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Biofilms, such as those formed by the opportunistic human pathogen Pseudomonas aeruginosa are complex, matrix enclosed, and surface-associated communities of cells. Bacteria that are part of a biofilm community are much more resistant to antibiotics and the host immune response than their free-floating counterparts. P. aeruginosa biofilms are associated with persistent and chronic infections in diseases such as cystic fibrosis and HIV-AIDS. P. aeruginosa synthesizes and secretes signaling molecules such as the Pseudomonas quinolone signal (PQS) which are implicated in quorum sensing (QS), where bacteria regulate gene expression based on population density. Processes such as biofilms formation and virulence are regulated by QS. This manuscript describes the powerful molecular imaging capabilities of confocal Raman microscopy (CRM) and surface enhanced Raman spectroscopy (SERS) in conjunction with multivariate statistical tools such as principal component analysis (PCA) for studying the spatiotemporal distribution of signaling molecules, secondary metabolites and virulence factors in biofilm communities of P. aeruginosa. Our observations reveal that the laboratory strain PAO1C synthesizes and secretes 2-alkyl-4-hydroxyquinoline N-oxides and 2-alkyl-4-hydroxyquinolones in high abundance, while the isogenic acyl homoserine lactone QS-deficient mutant (ΔlasIΔrhlI) strain produces predominantly 2-alkyl-quinolones during biofilm formation. This study underscores the use of CRM, along with traditional biological tools such as genetics, for studying the behavior of microbial communities at the molecular level.

Cytoskeletal networks are 3D arrangements of filaments whose complex spatial structure contributes significantly to their intracellular functions, e.g. biomechanics and cargo motility. Microtubule networks in cells are a particular challenge for in vitro modeling because they are sparse and possess overall structure and so cannot be approximated experimentally as a random hydrogel. We have used holographic optical trapping to precisely position and hold multiple microtubule filaments in an in vitro assay, where chemical and environmental variables can be carefully controlled. Below we describe the relevant practical details of the approach and demonstrate how our approach can scale to accommodate modeling of molecular motor transport and biomechanics experiments.

We show arrays of large gold nanodisks on glass substrates can support strong optical features with narrow spectral widths associated with their collective plasmonic-lattice modes. Our results show that these modes can offer significant sensitivity to the refractive index of the environment, far more than those of individual nanodisks. We show the visible collective modes supported by such arrays can distinctively detect a monolayer of biotin with high resolution. We use donor (CdSe/ZnS) and acceptor (CuInS/ZnS) quantum dots to investigate the field properties of these arrays after deposition of a thick layer of a silicon. We demonstrate a distinct increase of emission of CuInS/ZnS quantum dots, indicating the possibility of enhancement of energy transfer between these two types of quantum dots.

Semiconducting polymer dots (Pdots) are rapidly emerging fluorescent probes for bioanalysis. Pdots have extraordinarily strong absorption and bright emission compared to other commonly used fluorescent probes, making them very attractive for applications involving Förster resonance energy transfer (FRET). Here, we investigated two FRET systems with green-emitting poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) Pdots as donors and two different Cyanine 5 (Cy5) dyes as acceptors. A hydrophilic sulfo-Cy5 dye was directly conjugated to the Pdot surface using carbodiimide chemistry, and a hydrophobic Cy5 dye was observed to spontaneously partition into the core of the Pdot. FRET was observed to depend on the acceptor dye concentration with both systems, and was characterized using a combination of fluorescence emission spectra, excitation spectra, and lifetime measurements. Much stronger quenching of Pdot emission and FRET-sensitized acceptor dye emission were observed for the hydrophobic Cy5 system, and these trends were attributed to reduced donor-acceptor distances in comparison to the hydrophilic sulfo-Cy5 system. Current limitations in the experimental format are discussed. The results show that Pdots are effective FRET donors for acceptor dyes located both within and at the surface of Pdots.

We have engineered hybrid nanostructures derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETs), as they are capable of generating heat, reactive oxygen species (ROS) or emit fluorescence light. We present preliminary results that demonstrate the effectiveness of NETs for fluorescence imaging and photodynamic therapeutic destruction of breast cancer cells, upon photo-excitation using NIR light. These hybrid nanostructures present a promising platform with theranostic capability for future biomedical clinical applications.

Results show that nanoparticles (NPs) can be biosynthesized at room temperature on the reductive and chelating surfaces of Portobello mushroom spores (PMS). Using this green approach TiO_x, Ag, Au, Ag-TiO_x and Au-TiO_x NPs have been prepared. These were characterized by TEM, SIMS and μFTIR-FTIR. TiO_x/PMS, Ag-TiO_x/PMS, Au-TiO_x/PMS and Ag/PMS were active in bacterial inhibition towards Eschericia coli and Staphylococcus aureus, but Au/PMS was not active (suggesting a strong Au-PMS interaction). TiO_x/PMS, Ag/PMS and Ag-TiO_x/PMS were equally active in an antibacterial and an antifungal sense when tested against Asperillus and Candide. All samples (except Ag-TiO_x/PMS and Au-TiO_x/PMS) showed an interesting interaction with DNA. We report on the process of fine-tuning these antibacterial properties, progress on making these nanomaterials optically self-indicating and movement towards optical control of their antibacterial activity. Au-TiO_x/PMS shows a surface plasmon resonance (SPR) with a maximum at 518 nm that might be useful in following its anti-bacterial properties (i.e. making the bionanomaterial self-indicating). The future of such green bio-nanomaterials is strong.

Gold and silver nanoparticles exhibit unique optical properties in the visible range of the electromagnetic spectrum where the incident light excites coherent collective oscillations (plasmons) of conduction band electrons. The distance dependent near-field coupling between the nanoparticles leads to spectral shifts in the far-field, which makes plasmonic molecules unique distance sensors on the nanoscale. In general, two distinct coupling regimes can be differentiated in the near-field: the classical electromagnetic coupling regime and the quantum plasmonic coupling regime at very short interparticle separations. The plasmon driven charge transfer between nanoparticles in the quantum plasmonic regime is currently of high interest for developing new non-linear spectroscopies and sensors, but the role of molecules in the gap between the nanoparticles remains insufficiently understood. We investigated the impact of DNA on the transition from the classical coupling regime to the quantum plasmonic coupling regime. We found that for separations beyond approx. 2.8 nm, classical electromagnetic coupling dominates the spectral response of coupled nanoparticles. At shorter separations, the recorded spectra are significantly blue-shifted when compared to the classical prediction, indicative of quantum plasmonic effects. The presence of the DNA was crucial to sustain the spectral blue-shifts, indicating coherent charge transfer across the DNA as main cause for the effective depolarization of the Plasmon Ruler.

We show theoretically that when a semiconductor quantum dot and metallic nanoparticle system interacts with a laser field, quantum coherence can introduce a new landscape for the dynamics of Forster resonance energy transfer (FRET). We predict adsorption of biological molecules to such a hybrid system can trigger dramatic changes in the way energy is transferred, blocking FRET while the distance between the quantum dot and metallic nanoparticle (R) and other structural specifications remain unchanged. We study the impact of variation of R on the FRET rate in the presence of quantum coherence and its ultrafast decay, offering a characteristically different dependency than the standard 1/R⁶. Application of the results for quantum nanosensors is discussed.

Responsive or reactive materials offer the possibility for the development of low-power diagnostics for preventative healthcare. We have synthesized and characterized a functional polymeric material which irreversibly cleaves upon exposure to UV light. Because this cleavage is selective to UV wavelength, it could form the foundation of a UV indicator strip, allowing patients healthy and unhealthy populations to monitor their exposure. In a complementary project, we developed an all-fiber polarimetric elastography system for characterizing the mechanical properties of visco-elastic materials, such a tissue, and for correlating this signal with cellular/molecular-level markers.

Prosthetic grafts used for lower extremity revascularization and dialysis access fail due to hyperplastic stenosis and thrombosis. Graft surveillance is advocated to monitor function, however, graft failure can occur between episodic examinations. An innovative self-monitoring graft system allows automated surveillance with assessment of graft function using a cloud-based algorithm. We performed proof of concept experiments with in vitro and in vivo models to assess the feasibility such a real-time graft surveillance system. Initial in-vitro and in-vivo experiments demonstrate the ability for a self-monitoring graft system to remotely monitor hemodynamic parameters reflecting graft function using wireless data transmission. This automated system shows promise to deliver real-time data that can be analyzed by cloud-based algorithms alerting the clinician of a change in graft function or development of stenosis for further diagnostic study or intervention prior to graft failure.

Precise detection of trace amount of molecules, such as the disease biomarkers present in biofluids or explosive residues, requires high sensitivity detection. electrospray ionization–mass spectrometry (ESI-MS) is a common and effective technique for sensitive trace molecular detection in small-volume liquid samples. In ESI-MS, nano-liter volume samples are ionized and aerosolized by ESI, and fed into MS for mass analysis. ESI-MS has proven to be a reliable ionization technique for coupling liquid phase separations like liquid chromatography (LC) and capillary zone electrophoresis (CE) with the highly specific resolving power of MS. While CE and ESI can be performed on a microfluidic chip having a footprint of a few cm², MS is typically at least 100 times bigger in size than a micro-chip. A reduced size, weight, and power profile would enable semi-portable applications in forensics, environmental monitoring, defense, and biological/pharmaceutical applications. To achieve this goal, we present an initial study evaluating the use of mid-infrared absorption spectroscopy (MIRAS) in place of MS to create a ESI-MIRAS system. To establish feasibility, we perform ESI-MIRAS on phospholipid samples, which have been previously demonstrated to be separable by CE. Phospholipids are biomarkers of degenerative neurological, kidney, and bone diseases and can be found in biofluids such as blood, urine and cerebrospinal fluid. To establish sensitivity limits, calibration samples of 100 μM concentration are electrospray deposited on to a grounded Si wafer for different times (1 minutes to 4 minutes with a 1 minute step). The minimum detectable concentration-time product, where a FTIR globar is used as the MIR source, is found ~200 μM·s.

The ability to heal soft tissue injuries and regenerate cartilage is the Holy Grail of musculoskeletal medicine. Articular cartilage repair and regeneration is considered to be largely intractable due to the poor regenerative properties of this tissue. Due to their low self-repair ability, cartilage defects that result from joint injury, aging, or osteoarthritis, are the most often irreversible and are a major cause of joint pain and chronic disability. However, current methods do not perfectly restore hyaline cartilage and may lead to the apparition of fibro- or continue hypertrophic cartilage. The lack of efficient modalities of treatment has prompted research into tissue engineering combining stem cells, scaffold materials and environmental factors. The field of articular cartilage tissue engineering, which aims to repair, regenerate, and/or improve injured or diseased cartilage functionality, has evoked intense interest and holds great potential for improving cartilage therapy. Plasma-rich in growth factors (PRGF) and/or stem cells may be effective for tissue repair as well as cartilage regenerative processes. There is a great promise to advance current cartilage therapies toward achieving a consistently successful approach for addressing cartilage afflictions. Tissue engineering may be the best way to reach this objective via the use of stem cells, novel biologically inspired scaffolds and, emerging nanotechnology. In this paper, current and emergent approach in the field of cartilage tissue engineering is presented for specific application. In the next years, the development of new strategies using stem cells, in scaffolds, with supplementation of culture medium could improve the quality of new formed cartilage.

Ultra-high-molecular-weight polyethylene (UHMWPE, PE) is a synthetic polymer used for biomedical applications because of its high impact resistance, ductility and stability in contact with physiological fluids. Therefore this material is being used in human orthopedic implants such as total joint replacements. Surface modification of this material relates to changes of its surface hydrophilicity, energy, microstructure, roughness, and morphology, all influencing its biological response.

In our recent work, PE was treated by an Ar⁺ plasma discharge and then grafted with biologically active polyethylene glycol in order to enhance adhesion and proliferation of mouse fibroblast (L929). The surface properties of pristine PE and its grafted counterparts were studied by goniometry (surface wettability). Furthermore, Atomic Force Microscopy was used to determine the surface morphology and roughness. The biological response of the L929 cell lines seeded on untreated and plasma treated PE matrices was quantified in terms of the cell adhesion, density, and metabolic activity.

Plasma treatment leads to the ablation of the polymer surface layers. Plasma treatment and subsequent poly(ethylene glycol) grafting lead to dramatic changes in the polymer surface morphology and roughness. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with poly(ethylene glycol) increases cell proliferation compared to plasma treatment.

The rapid advancement in technology has envisaged and drafted the use of optical bio-sensing units into label free and multiplexed bio-sensing, exploring the surface plasmon polaritons, which has turned into a gold standard on the commercial basis, but they are bulky and find difficulty in scaling up for the throughput detection. The integration of plasmonic crystals with microfluidics on the bio-sensing frontier offers a multi-level validation of results with the ease of real-time detection and imaging and holds a great promise to develop ultra-sensitive, fast, portable device for the point-of-care diagnostics. The paper describes the fast, low cost approach of designing and simulating label free biosensor using open source MEEP and other software tools targeting Immunodiagnostics.

In this study, we proposed a novel method to obtain fine fibers: electrohydrodynamic (EHD) direct printing PCL on hydrogel. The effects of applied voltage, flow rate, plotting speed, hydrogel viscosity on the EHD direct writing process were investigated to obtain the most appropriate set of process conditions. We also compared the fibers obtained from this method with traditional EHD which deposited on aluminum and ethanol-based collectors. We found that fibers collected on the hydrogel were thinner and exhibited pores on the surface due to the hydrogel’s facilitation of organic solvents’ volatilization in the PCL fibers, which can benefit the attachment of cells. Besides, in our previous study, we found the freeze-thaw crosslinking process could greatly increase mechanical properties of the hydrogel used in our research, so this integration will not only mimic the composition of ECM which is a composite structure with a combination of fibrous proteins within a gelatinous grounded substance but also improve the mechanical properties of the scaffold. This novel method will broaden the application of EHD technology in the field of tissue engineering and other related areas.