Single-molecule Detection in Nanogap-embedded Plasmonic Gratings

We introduce nanogap-embedded silver plasmonic gratings for single-molecule (SM) visualization using an epifluorescence microscope. This silver plasmonic platform was fabricated by a cost-effective nano-imprint lithography technique, using an HD DVD template. DNA/RNA duplex molecules tagged with Cy3/Cy5 fluorophores were immobilized on SiO2-capped silver gratings. Light was coupled to the gratings at particular wavelengths and incident angles to form surface plasmons. The SM fluorescence intensity of the fluorophores at the nanogaps showed approximately a 100-fold mean enhancement with respect to the fluorophores observed on quartz slides using an epifluorescence microscope. This high level of enhancement was due to the concentration of surface plasmons at the nanogaps. When nanogaps imaged with epifluorescence mode were compared to quartz imaged using total internal reflection fluorescence (TIRF) microscopy, more than a 30-fold mean enhancement was obtained. Due to the SM fluorescence enhancement of plasmonic gratings and the correspondingly high emission intensity, the required laser power can be reduced, resulting in a prolonged detection time prior to photobleaching. This simple platform was able to perform SM studies with a low-cost epifluorescence apparatus, instead of the more expensive TIRF or confocal microscopes, which would enable SM analysis to take place in most scientific laboratories

Compact shock wave generating device for drug delivery

Genetic Engineering and Biotechnology News reported that "the burgeoning markets that surround biopharmaceuticals, RNA interference screening, and stem cell research are limited by the lack of a silver bullet for successful gene transfer. Because stable transfection is hard to achieve in primary cell lines, this application continues to be an important untapped niche in the transfection market." Inventors developed a microdevice creating shock waves from the reaction of nanoenergetic materials of fuel and oxidizer in nanoscale. The shock waves then permeabilize target cells allowing delivery of genetic material into the cells. The characteristics of the shock wave that can be controlled include pulse intensity, and pulse duration. The tunability of the shock waves allows the device to be adapted for use in a wide range of applications. DNA and nanoparticle delivery have been demonstrated. As compared to existing cell transfection products, this device achieves a significantly greater transfection success rate, significantly greater cell survival rate, and should cost less than most, if not all other methods. The invention was compared with commercially available chemical-based transfections (SiPort NeoFx, SiPort Amine, Lipofectamine 2000, Lipofectamine LTX, Transit LT1), and electroporation. The prototype of the invention produced transfection and survivability rates in excess of 99% while none of the existing transfection methods resulted in a rate greater than 10%, and the survivability of those transfected cells ranged from 0% to 80%. This device has the potential to revolutionize cell transfection, as the shock waves are particularly good at making the cell membranes porous, while at the same time the shock waves are gentle and do not cause catastrophic damage during the transfection, so cells survive. Potential Areas of Applications: * Cell Transfection * Shockwave drug delivery for killing cancer cells * Precision drug delivery of imaging particles * Fragmentation of kidney stones * Destruction of plaques Patent Status: Prototype tested and patent application 12/253,706 published Inventor(s): Dr. Shubhra Gangopadhyay, Ph.D.; Dr. Steven Apperson, Ph.D.; Dr. Luis Polo-Parada, Ph.D.; Dr. Keshab Gangopadhyay, Ph.D.; Dr. Andrey Bezmelnitsyn, Ph.D. Contact Info: Dr. Wayne McDaniel, Ph.D. ; [email protected] ; 573-884-330

Design and development of nanoenergetic materials with tunable combustion characteristics [abstract]

In recent years, nanoengineered thermites with tunable and tailored characteristics have attracted a great deal of attention owing to their enormous potential as excellent reactive materials, green primers, and structural energetic materials etc. Nanothermites are typically composed of metal oxide (oxidizer) and metal (fuel) nanoparticles. A variety of nanostructured oxidizers such as Fe2O3, CuO, Bi2O3 and MoO3 etc have been prepared in our laboratory. Various morphologies of oxidizers include nanorods, nanoparticles, and mesoporous structures exhibiting high surface area. Surfactant templating method has been developed for the synthesis of ammonium nitrate (NH4NO3) nanoparticles with a size distribution of 10-100nm. The physical and the chemical properties such as morphology, surface area, purity, composition, crystal structure of these metal oxide nanostructures have been determined by a host of characterization tools. Among the nanothermites, CuO nanorods/Al nanoparticles exhibit the best combustion performance measured in terms of combustion wave speed of 2600 100 m/s and reactivity of 11 1 MPa/msec. Nanothermites based on CuO nanorods/Al nanoparticles were then modified by mixing with polymers such as nitrocellulose (NC) and/or explosives such as (NH4NO3) nanoparticles, RDX (micron and nano size) and CL20 and the reaction rates of these nanocomposites were determined. Among the polymers, nitrocellulose coating of nanothermites is very interesting. Both the NC and the Teflon coated CuO/Al based nanothermite systems exhibit the ability to generate shock waves during their fast combustion. The NC coating has shown tremendous potential to reduce the high sensitivity of nanothermites to electrostatic discharge (ESD), friction and impact. Experimentally measured combustion characteristics are found to correlate very well with the physical and chemical characteristics of metal oxide nanostructures. The developed technology in our lab demonstrates the potential to tune and tailor the combustion characteristics of nanothermites to the desired level by proper choice and combination of fuel and oxidizer materials, their dimensions, and the process of self-assembly with reduced sensitivity. Potential Areas of Applications: * Microthrusters; * Propellants; * Propellant Initiators; * Suitable Replacements for Lead and Sulfur based Primers; * Shockwave drug delivery system

Entropy driven spontaneous formation of highly porous films from polymer-nanoparticle composites

doi: 10.1088/0957-4484/20/42/425602Nanoporous materials have become indispensable in many fields ranging from photonics, catalysis and semiconductor processing to biosensor infrastructure. Rapid and energy efficient process fabrication of these materials is, however, nontrivial. In this communication, we describe a simple method for the rapid fabrication of these materials from colloidal dispersions of Polymethyl Silsesquioxane nanoparticles. Nanoparticle-polymer composites above the decomposition temperature of the polymer are examined and the entropic gain experienced by the nanoparticles in this rubric is harnessed to fabricate novel highly porous films composed of nanoparticles. Optically smooth, hydrophobic films with low refractive indices (as low as 1.048) and high surface areas (as high as 1325 m2 g−1) have been achieved with this approach. In this communication we address the behavior of such systems that are both temperature and substrate surface energy dependent. The method is applicable, in principle, to a variety of nanoparticle-polymer systems to fabricate custom nanoporous materials.We gratefully acknowledge the financial support from National Institute of Health (Award number 2-U42RR014821) and the US army

Novel Nanostructured Organosilicate Nanoparticle Coatings for Chem-Bio Sensing [abstract]

Plenary speakerWe present novel nanostructured organosilicate particulate based films and demonstrate that these materials have a great potential for chemical-biological sensor development. With unprecedented high surface areas (> 1400 m2/g) and optical transparency together with its easy surface functionalization, these materials can be readily interfaced with existing immunoassays for the rapid and trace detection of both chemical and biological warfare agents. The ultra high surface area associated with these films stems from its unique nanostructure consisting of nanoparticles (2-5nm) in a “raspberry” structure in combination with interconnected nanopores (3-10nm). This unique nanostructure has been exploited to immobilize high areal density of sensor probes to improve the sensing performance. Two orders of magnitude increase in binding density was achieved when fluorescently tagged protein A molecules were immobilized upon these surfaces compared to flat substrates (glass and Silicon). Our on-going work applies these materials to develop platforms for multiplexed sensitive detection of biological and chemical agents at point of care for both army and civilian use

Development and characterization of fluorescent dye-doped nanoparticles with enhanced fluorescence intensity and photostability [abstract]

Nanoscience Poster SessionWe report the development of fluorescent dye doped organosilicate nanoparticles (DOSNPs) synthesized from poly-methylsilsesquioxane(PMSSQ), resulting in high fluorescence intensity and excellent photostability. The surface modified DOSNPs have hydrophilic surfaces and hydrophobic cores that enhance water-solubility and protect the dyes from oxidation and phtobleaching. These DOSNPs show superior properties over conventional dyes such as high fluorescence intensity due to approximately hundred dye molecules per particle and photostability demonstrating 7% and 76% fluorescence decay under continuous excitation for rhodamine 6G (R6G) DOSNP and R6G molecules, respectively, and have potential to be used in many areas, for example, imaging, sensing and solar cells. DOSNPs, when conjugated to anti-fibronectin antibodies, increased sensitivity of detection by approximately 600 fold relative to individual dye molecules conjugated to antibody. The DOSNPs are being applied to the development of diagnostic devices to be used in the detection of drugs, metabolites and pathogens

Organosilicate nanoparticles and its applications in chem-biosensors, electronics, multifunctional coatings and textiles

This invention reports a novel technique for the rapid and cost-efficient synthesis of organosilicate nanoparticles (OSNPs) that have been successfully applied as individual building blocks for various applications. Doping these nanoparticles with fluorescent dyes results in highly fluorescent, biocompatible, water soluble nanoparticles with demonstrated long term photostability and with surface groups that can be readily used to attach various biological moieties. Fluorescent intensity of dye doped OSNPs (22.4 ± 5.3 nm) is shown to be 200 times brighter with 94% of the initial fluorescence intensity retained than the constituent dyes under continuous excitation for 10 minutes. In contrast, under identical test conditions, individual dye molecules retained only 58% of the initial fluorescence demonstrating that these nanoparticles have excellent utility in lifesciences research, forensics, chemical - biological sensors and biological imaging applications. Through our patented technology of novel bottom up fabrication technique, these nanoparticles have been used to fabricate highly porous transparent films. Optically smooth hydrophobic films with low refractive indices (as low as 1.048) and high surface areas (as high as 1325 m2/g) can be achieved on large area substrates. These unique materials can be readily interfaced with existing immunoassays in the form of inexpensive dip-stick assays for the sensitive detection of chemical and biological warfare agents or novel diagnostic strips for point of care applications. Our preliminary evaluation of these coatings in combination with dye doped OSNPs for construction of diagnostic immunoassays gave ~180 fold enhancement in fluorescence signal enhancement compared to traditional (microscope glass slide and fluorescent dye molecules) based assays. OSNPs used as filler elements within sol-gel based coatings have been shown to greatly enhance their structural stability, flexibility and wear resistance. Crack-free coatings (with thicknesses exceeding 30 microns)/novel multifunctional electrospun fibers have been successfully achieved by employing OSNP fillers (up to 75% by weight) within sol-gel compositions. POTENTIAL AREAS OF APPLICATIONS: *Chemical Biological sensors *Medical Diagnostics *Multifunctional coatings *Next generation Chemical-Biological protection textiles (Soldier technologies) PATENT STATUS: Non provisional patent application on file INVENTOR(S): Sangho Bok; Venumadhav Korampally; Luis Polo-Parada; Vamsi Mamidi; Keshab Gangopadhyay; William R. Folk; Purnendu K. Dasgupta and Shubhra Gangopadhyay CONTACT INFO: Wayne McDaniel, Ph.D.; [email protected] ; 573-884-330

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively

Generation of fast propagating combustion and shock waves with copper oxide/aluminum nanothermite composites

doi:10.1063/1.2787972Nanothermite composites containing metallic fuel and inorganic oxidizer are gaining importance due to their outstanding combustion characteristics. In this paper, the combustion behaviors of copper oxide/aluminum nanothermites are discussed. CuO nanorods were synthesized using the surfactant-templating method, then mixed or self-assembled with Al nanoparticles. This nanoscale mixing resulted in a large interfacial contact area between fuel and oxidizer. As a result, the reaction of the low density nanothermite composite leads to a fast propagating combustion, generating shock waves with Mach numbers up to 3.We gratefully acknowledge the financial support by U.S. Army, ARDEC, New Jersey and National Science Foundation

Shock Wave Based Cell Transfection and Fluorescent Organosilicate Nanoparticles for Targeted Drug Delivery [abstract]

Nanoscience Poster SessionNanotechnology is a multidisciplinary field that has applications in life sciences, alternative energy, national defense, and electronics. In the field of medicine, nanotechnology may enable intelligent drug delivery using multifunctional nanoparticles. Here, we show two technologies that are envisioned to work in tandem to enable targeted detection and treatment. First, a shock wave generator used for cell transfection and drug/particle delivery is presented. Then, fluorescent dye/drug encapsulated organosilicate nanoparticles (OSNP) with functionalized surfaces for targeted delivery are described. The shock wave generator has been successfully used to deliver various molecules and nanoparticle to inside of the cells with very high efficiency and low cell damage. These include dextran (77 kDa), naked plasmid, and dye-doped organosilicate nanoparticles into several types of cells lines including T47-D, HL-60, and MCF-7, and also into tissues including entire chicken heart (at developmental stage 20-30) and chicken spinal cord. Dye doped organosilicate nanoparticle surfaces conjugated to antibodies have been successfully used in immunofluorescence assays. Close examination of the nanostructure of these particles reveal its unique nanoporous structure. These nanoparticles are currently under investigation for drug encapsulation and sustained release. The implication of these technologies is that the OSNP can be used as targeted drug carriers, and the shock wave generator can be used to deliver the OSNP into cells to which the particles attach. The research on shock wave micro-transfector system has been funded by the National Science Foundation Grant Opportunities for Academic Liason with Industries program