Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immunotherapy-Relevant Antibody-Ligand Interactions.

We fabricated a novel single molecule nanosensor by integrating a solid-state nanopore and a double nanohole nanoaperture. The nanosensor employs Self-Induced Back-Action (SIBA) for optical trapping and enables SIBA-Actuated Nanopore Electrophoresis (SANE) for concurrent acquisition of bimodal optical and electrical signatures of molecular interactions. This work describes how to fabricate and use the SANE sensor to quantify antibody-ligand interactions. We describe how to analyze the bimodal optical-electrical data to improve upon the discrimination of antibody and ligand versus bound complex compared to electrical measurements alone. Example results for specific interaction detection are described for T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting Major Histocompatibility Complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells. We also describe how to analyze the bimodal optical-electrical data to discriminate between specific and non-specific interactions between antibodies and ligands. Example results for non-specific interactions are shown for cancer-irrelevant TCRmAbs targeting the same pMHCs, as a control. These example results demonstrate the utility of the SANE sensor as a potential screening tool for ligand targets in cancer immunotherapy, though we believe that its potential uses are much broader.

Detection of specific antibody-ligand interactions with a self-induced back-action actuated nanopore electrophoresis sensor.

Recent advances in plasmonic nanopore technologies have enabled the use of concurrently acquired bimodal optical-electrical data for improved quantification of molecular interactions. This work presents the use of a new plasmonic nanosensor employing self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) for quantifying antibody-ligand interactions. T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting major histocompatibility complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells, were used to test the SANE sensor's ability to identify specific antibody-ligand binding. Cancer-irrelevant TCRmAbs targeting the same pMHCs were also tested as a control. It was found that the sensor could provide bimodal molecular signatures that could differentiate between antibody, ligand and the complexes that they formed, as well as distinguish between specific and non-specific interactions. Furthermore, the results suggested an interesting phenomenon of increased antibody-ligand complex bound fraction detected by the SANE sensor compared to that expected for corresponding bulk solution concentrations. A possible physical mechanism and potential advantages for the sensor's ability to augment complex formation near its active sensing volume at concentrations lower than the free solution equilibrium binding constant (K D ) are discussed.

Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics.

Ovarian carcinoma has the highest lethality rate of gynecologic tumors, largely attributed to the late-stage diagnosis of the disease. Reliable tools for both accurate diagnosis and early detection of disease onset are lacking, and presently less than 20% of ovarian cancers are detected at an early stage. Protein biomarkers that allow the discrimination of early and late stages of ovarian serous carcinomas are urgently needed as they would enable monitoring pre-symptomatic aspects of the disease, disease progression, and the efficacy of intervention therapies. We compare the absolute and relative protein levels of six protein biomarkers for ovarian cancer in five different established ovarian cancer cell lines, utilizing both quantitative immunoblot analysis and a guided-mode resonance (GMR) bioassay detection system that utilizes a label-free optical biosensor readout. The GMR sensor approach provided highly accurate, consistent, and reproducible quantification of protein biomarkers as validated by quantitative immunoblotting, as well as enhanced sensitivity, and is therefore suitable for quantification and detection of novel biomarkers for ovarian cancer. We identified fibronectin, apolipoprotein A1, and TIMP3 as potential protein biomarkers for the differential diagnosis of primary versus metastatic ovarian carcinoma. Future studies are needed to confirm the suitability of protein biomarkers tested herein in patient samples.

Resonant photonic biosensors with polarization-based multiparametric discrimination in each channel.

In this paper, we describe guided-mode resonance biochemical sensor technology. We briefly discuss sensor fabrication and show measured binding dynamics for example biomaterials in use in our laboratories. We then turn our attention to a particularly powerful attribute of this technology not possessed by competing methods. This attribute is the facile generation of multiple resonance peaks at an identical physical location on the sensor surface. These peaks respond uniquely to the biomolecular event, thereby enriching the data set available for event quantification. The peaks result from individual, polarization-dependent resonant leaky modes that are the foundation of this technology. Thus, by modeling the binding event and fitting to a rigorous electromagnetic formalism, we can determine individual attributes of the biolayer and its surroundings and avoid a separate reference site for background monitoring. Examples provide dual-polarization quantification of biotin binding to a silane-coated sensor as well as binding of the cancer biomarker protein calreticulin to its monoclonal IgG capture antibody. Finally, we present dual-polarization resonance response for poly (allylamine hydrochloride) binding to the sensor with corresponding results of backfitting to a simple model; this differentiates the contributions from biolayer adhesion and background changes.

Bacterial biofilm formation on a human cochlear implant.

OBJECTIVE: To report the characteristics of a bacterial biofilm from the surface of a cochlear implant. BACKGROUND: Bacterial biofilm formation on implanted devices causes intractable infections and device extrusions necessitating device removal, with loss of function. More information is needed about biofilm characteristics and interactions with the implant surface before better treatments can be designed. STUDY DESIGN: A retrospective case review was combined with a descriptive histological study of the surface of an otologic device. METHODS: The receiver/stimulator device removed from a cochlear implant patient because of intractable infection and partial device extrusion was fixed and processed for microscopic examination. Its surface and the material present on its surface were analyzed using light and electron microscopy, focusing on surface texture, cell types, and bacteria species and extracellular polymeric substances present within the biofilm. RESULTS: Stereomicroscopic examination revealed extracellular polymeric substances, pinkish yellow in color, with spheres of uniform size scattered throughout, indicative of a biofilm containing Staphylococcus aureus. Biofilm density was greatest in depressions on the surface of the implant. Cross-sectional analysis revealed bacteria interspersed with polymorphonuclear leukocytes. Scanning electron microscopic examination demonstrated an amorphous layer of extracellular polymeric substances containing small filaments, bacteria, and inflammatory cells. Only Staphylococcus aureus was detected. CONCLUSION: Cochlear implant material can provide a surface for bacterial biofilm formation. Impressions can provide an environment conducive to biofilm establishment and growth, ultimately necessitating device removal, with loss of implant function. Biofilm characterization should aid in design of cochlear implant devices less susceptible to biofilm formation.

Molecular responses of vascular smooth muscle cells to paclitaxel-eluting bioresorbable stent materials.

We studied the influence of paclitaxel, eluted from poly(L-lactic acid) (PLLA), on cultured vascular smooth muscle cell (VSMC) proliferation as a model of bioresorbable stent-induced restenosis. We blended paclitaxel in cast PLLA films (P-PLLA), demonstrating controlled release of the drug, then studied VSMC adhesion, proliferation, and gene expression profiles. No difference in cell adhesion was found between P-PLLA and PLLA controls (105 +/- 12% of PLLA controls). However, P-PLLA significantly reduced VSMC proliferation (40 +/- 15% of PLLA controls, p < 0.05). Using cDNA microarray technology, we identified major effects of P-PLLA, including: upregulation of genes related to apoptosis, anti-proliferation and antioxidation; and suppression of cell cycle regulators and cell survival markers. The expression patterns indicate that P-PLLA regulates gene expression and cell functions via new pathways, including receptor tyrosine kinase (RTKs), mitogen-activated protein kinase (MAPKs), and protein kinase (PKs, e.g., PKA) pathways, in addition to the stabilization of polymerized-microtubules.