This PDF file contains the front matter associated with SPIE Proceedings Volume 7759, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

In order to enhance the sensitivity of conventional rapid test technique for the detection of swine-origin influenza A (H1N1) viruses (S-OIVs), we used a paired surface plasma waves biosensor (PSPWB) based on SPR in conjunction with an optical heterodyne technique. Experimentally, PSPWB showed a 125-fold improvement at least in the S-OIV detection as compared to conventional enzyme linked immunosorbent assay. Moreover, the detection limit of the PSPWB for the S-OIV detection was enhanced 250-fold in buffer at least in comparison with that of conventional rapid influenza diagnostic test.

We present the design and initial characterization of a device geometry that is suitable for measuring the direct midinfrared absorption of single molecules. The devices are based on a metal-dielectric-metal sandwich with optically thick gold cladding layers which squeeze the gap mode, the optical mode in the dielectric layer, in the electric field direction; with a photonic crystal cavity defect which can squeeze the gap mode in the planar direction. Simulations show a field enhancement of 10000 times the incident field in the cavity defect at an excitation frequency of 87 THz. Experimentally, devices with varying periods were made using a free standing Si₃N₄ film of 15 nm or 50 nm as the inner dielectric. These devices show a red shift as the period is increased and more interesting there is also a red shift for the thinner dielectric devices showing that the field is further squeezed in this layer. By placing a molecule into the cavity, there should be a strong enough interaction between the light and the analyte so that its absorption spectra can be resolved.

We developed a wafer-scale 3D plasmonic crystal consisting of gold nanopyramid arrays using a colloidal templating technique. These surface plasmons in the arrays allow us to obtain real-time, label-free and high sensitivity biomolecular binding measurements. In our research, we found the sensing capabilities improved while the detection limit of the alcohol dehydrogenase sensing was down to pM. The features of low cost fabrication and real-time, label-free, specific and quantitative measurement capabilities suggest promise for the gold nanopyramid arrays in both biological and chemically based analytical detection systems.

An enzyme based biosensor was fabricated by employing a simple, inexpensive and rapid xurography fabrication process. The electrodes and channel were made from the conducting polymer poly(3,4-ethyelenedioxythiphene) poly(styrene sulfonate) (PEDOT:PSS). PEDOT:PSS was selectively deposited using a polyimide tape mask. The tape mask was peeled off from the substrate after annealing the polymer in vacuum. Polymer wells of defined dimensions were made and were attached to the device to accommodate the solutions. This sensor utilizes the change in current as a parameter to measure different analyte concentrations. Initial experiments were done by using the sensor for glucose detection. The sensor is able to detect the glucose concentrations approximately from 1 μM to 10 mM range covering glucose in human saliva (8-210 μM). The glucose oxidase activity was independently measured using colorimetric method and the results indicate that the sensor retains the enzyme activity and can be used as a biosensor to detect various analytes. The analyte of interest can be measured by preloading the corresponding enzyme into the wells.

The Army has a need for an accurate, fast, reliable and robust means to identify and quantify defense related materials. Raman spectroscopy is a form of vibrational spectroscopy that is rapidly becoming a valuable tool for homeland defense applications, as it is well suited for the molecular identification of a variety of compounds, including explosives and chemical and biological hazards. To measure trace levels of these types of materials, surface enhanced Raman scattering (SERS), a specialized form of Raman scattering, can be employed. The SERS enhancements are produced on, or in close proximity to, a nanoscale roughened metal surface and are typically associated with increased local electromagnetic field strengths. However, before application of SERS in the field and in particular to biological and other hazard sensing applications, significant improvements in substrate performance are needed. In this work, we will report the use of several SERS substrate architectures (colloids, film-over-nanospheres (FONs) and commercially available substrates) for detecting and differentiating numerous endospore samples. The variance in spectra as obtained using different sensing architectures will also be discussed. Additionally, the feasibility of using a modified substrate architecture that is tailored with molecular recognition probe system for detecting biological samples will be explored. We will discuss the progress towards an advanced, hybrid molecular recognition with a SERS/Fluorescence nanoprobe system including the optimization, fabrication, and spectroscopic analysis of samples on a commercially available substrate. Additionally, the feasibility of using this single-step switching architecture for hazard material detection will also be explored.

In this study, we describe a novel method for analyzing protein-protein binding kinetics at ultra-low concentration (1 pg/mL) using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). The association and dissociation rate constants, ka and kd, respectively, for the binding kinetics of the mouse IgG/ anti-mouse IgG interaction have been calculated to be ka = (5.9928±3.1540)x10⁶ M^-1s^-1 and kd = (1.0587±0.5572)x10^-3 s^-1. The theoretical basis of this analytical approach is a rapid-mixing model integrated with a two-compartment model; has been experimentally verified in this study as well. The LSPCF-FOB provides a potentially alternative option for characterizing the interaction of biomolecules at ultra-low concentrations.

Surface Plasmon Resonance imaging (SPRi) is a label-free technique for the quantitation of binding affinities and concentrations for a wide variety of target molecules. Although SPRi is capable of determining binding constants for multiple ligands in parallel, current commercial instruments are limited to a single analyte stream and a limited number of ligand spots. We have developed an integrated microfluidic array using soft lithography techniques for SPRi-based detection and determination of binding affinities for DNA aptamers against human alpha-thrombin. The device consists of 264 element-addressable chambers of 700 pL each isolated by microvalves. The device also contains a dilution network for simultaneous interrogation of up to six different target concentrations, further speeding detection times. The element-addressable design of the array allows interrogation of multiple ligands against multiple targets, and analytes from individual chambers may be collected for downstream analysis. We demonstrate methods for educing nonspecific binding to the sensor surface and quantify the success of these methods using mass spectrometric identification of proteins eluted from our microfluidic chambers following SPRi analysis of crude cell lysates.

Inertial components of the Navier-Stokes equations are usually not considered in microfluidic flows but have recently been shown to be of great practical use for continuous manipulation of particles and cells. After introducing the physical basis of the counter-intuitive self focusing of particles in a single inlet flow, I will discuss our current best focusing systems, and I will present results on using inertial focusing to create an extreme throughput flow cytometer for blood analysis. This system is an imaging cytometer implementation that can image 1 million focused blood cells per second, with the capability to increase to 20 million cells per second with appropriate wide-field of view imaging systems. The microfluidic device consists of 256 parallel high-aspect ratio microchannels in each of which two streams of focused cells assemble. These cells also form regular trains in the direction of flow such that cell coincidence is a rare occurrence, far below Poisson statistics suggest. Controlled inertially focused streams of particles are poised to provide next-generation filter-less filters and simplified flow cytometry instruments which ultimately may aid in water treatment environmental cleanup and cost-effective medical diagnostics.

The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 10⁹ particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

In this report we propose a new instrumental method for the biochemical diagnostics of the bovine leucosis through the registration of the formation of the specific immune complex (antigen-antibody) with the help of biosensor based on the nano-structured silicon. The principle of the measurements is based on the determination of the photosensitivity of the surface. In spite of the existed traditional methods of the biochemical diagnostics of the bovine leucosis the proposed approach may provide the express control of the milk quality as direct on the farm and during the process raw materials. The proposed variant of the biosensor based on the nano-structured silicon may be applied for the determination of the concentration of different substances which may form the specific complex in the result of the bioaffine reactions. A new immune technique based on the nanostructured silicon and intended for the quantitative determination of some toxic substances is offered. The sensitivity of such biosensor allows determining T-2 mycotoxin at the concentration of 10 ng/ml during several minutes.

In this paper we present an overview of our work on a method to provide three-dimensional (3D) identification and tracking of biological micro/nano-organisms. This approach connects digital holographic microscopy and statistical methods for cell identification. For 3D data acquisition of living biological microorganisms, a filtered white light source, LED or laser diode beam propagates through a biological microorganism and the transversely and longitudinally magnified Gabor hologram pattern of the biological microorganism by microscope objective is optically recorded with a CCD camera interfaced with a computer. 3D imaging of the biological microorganism from the magnified Gabor hologram pattern is obtained by applying the computational Fresnel propagation algorithm. For identification and tracking of the biological microorganism, statistical approaches based on statistical estimation and inference algorithms are developed to the segmented holographic 3D image. Overviews of analytical frameworks are discussed and experimental results are presented.

The forces exerted by an adherent cell on a substrate were studied previously only in the two-dimensions (2D) tangential to the substrate surface. We used a novel technique to measure the three-dimensional (3D) stresses exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate, with particular emphasis on the 3D forces of focal adhesions. On 3D images acquired by confocal microscopy, displacements were determined with imageprocessing programs, and stresses in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated stress in normal direction (Tz) with an order of magnitude comparable to that in tangential direction (Txy). Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. With the use of green fluorescent protein (GFP) labeled paxillin, the dynamics of this intracellular molecule were studied concurrently with the measurement of 3D forces. In the dynamic region, including the new lamellapodium forming region in the front and the retracting region in the rear, the tangential forces (Fxy) are correlated with the size of the focal adhesions (FAs) much more strongly than those in the stable region under the nucleus. In the dynamic region, normal force (Fz) was upward and positively correlated with FA size, while Fz in the stable region was downward and negatively correlated with FA size. These findings show the influence of the size of FAs on the 3D forces they exert on the substrate. This technique can be applied to study any adherent type of live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, thus elucidating cellular functions in health and disease.

We develop second-harmonic nanoparticles as the contrast agents for cell imaging. Second-harmonic nanoparticles show promise as cell imaging probes due to their non-bleaching, non-blinking, and coherent signal. Nanoparticles of noncentrosymmetric crystal structures have high second-harmonic generation (SHG) efficiency and provide high contrast in a generally non-structured cell environment. Here, we use barium titanate (BaTiO₃) nanoparticles with tetragonal crystal structure as imaging probes. Cytotoxicity tests performed on BaTiO₃ nanoparticles with mammalian cells did not result in toxic effects. Specifically, we observed no change in the cell metabolism after 24 hours incubation of the cells with high concentration of BaTiO₃ nanoparticles. We demonstrate two methods of cell labeling with BaTiO₃ nanoparticles for imaging. One is non-specific labeling via endocytosis of the cells, which results in a great number of the nanoparticles randomly distributed inside the cells. The other is specific labeling via surface functionalization of the nanoparticles with antibodies, which enables us to label specific cell membrane proteins with the nanoparticles. SHG imaging is compatible to two-photon microscopy and the SHG signal from nanoparticles can be easily detected with a standard two-photon confocal microscope. Our work provides the opportunity for long-term, three-dimensional cell tracking with secondharmonic nanoparticles.

We describe a microfluidic device capable of trapping, isolating, and lysing individual cells in parallel using dielectrophoretic forces and a system of PDMS channels and valves. The device consists of a glass substrate patterned with electrodes and two PDMS layers comprising of the microfluidic channels and valve control channels. Individual cells are captured by positive dielectrophoresis using the microfabricated electrode pairs. The cells are then isolated into nanoliter compartments using pneumatically actuated PDMS valves. Following isolation, the cells are lysed open by applying an electric field using the same electrode pairs. With the ability to capture and compartmentalize single cells our device may be combined with analytical methods for in situ molecular analysis of cellular components from single cells in a highly parallel manner.

We report on a novel fluid actuation mechanism capable of achieving ultrafast actuation in micro and nanofluidic environment by utilizing laser pulse induced cavitation bubbles. A highly focused laser beam can initiate a vapor bubble with a rapidly expansion speed at more than 100 m/sec in few nanoseconds and an internal pressure of tens of MPa. Such a fast response and large force induced laser triggered cavitation bubble could strongly perturb the fluid flow near the focal point and the neighboring channel structures. We have demonstrated several novel applications based on this mechanism, including a high speed microfluid membrane switch with a cycle lifetime less than 6 μsec, and a high speed droplet device capable of generating highly uniform droplet at a speed of 10,000 droplets per second on demand.

In this work, we show the use of single stranded DNA aptamers as selective biorecognition elements in a sensor based on a field effect transistor (FET) platform. Aptamers are chemically attached to the semiconducting material in the FET through the use of linker molecules and confirmed through atomic force microscopy and positive target detection. Highly selective sensing of a small molecule, riboflavin is shown down to the nano-molar level in zinc oxide FET and micro-molar level in a carbon nanotube FET. High selectivity is determined through the use of negative control target molecules with similar molecular structures as the positive control targets with little to no sensor response. The goal of this work is to develop a sensor platform where biorecognition elements can be used to functionalize an array of transistors for simultaneous sensing of multiple targets in biological fluids.

DNA hybridization can be measured with enhanced sensitivity based on localized surface plasmon (LSP) induced by surface nanowire structure. Changes made to the structure result in higher plasmon momentum, which can be coupled to a particle plasmon induced by gold nanoparticles to which DNA molecules are adsorbed. With the insight gained from near-field pattern via calculation, target localization effect is also experimentally shown. We expect that orders of magnitude can be improved in terms of sensitivity if one is to combine the effect of particle-to-LSP coupling and target localization scheme.

Various nanoparticles play a prominent role in modern biosciences and medicine. Especially fluorescent cellular biomarkers are a prospective material for diagnostics and therapy. Nevertheless, most of the available biomarkers have some drawbacks due to either physical and optical or cytotoxic properties. Here we investigated the potential of green fluorescent nanodiamonds as extra- and intracellular biomarkers for living cells. We characterized the structure of the used detonation synthesized nanodiamonds (DND) by X-ray diffraction (XRD) and the optical properties by fluorescence and infrared spectroscopy. For the extracellular attachment the nanodiamonds were functionalized by attaching antibodies that target extracellular structures such as membrane. Transfections were mediated by dendrimers, cationic liposomes and protamine sulfate. Using fluorescence microscopy, we confirmed successful extracellular binding on and transfection of the nanodiamonds into prostate cancer cells. Furthermore, nanodiamonds can be targeted selectively to intracellular structures. Therefore, nanodiamonds are a promising tool for biosensing.

In this work, a nanoscale surface-enhanced Raman scattering (SERS) substrate is fabricated by fs laser reduction and deposition. The conductive silver microstructures are also deposited in fs laser irradiated area on the glass surfaces. Based on this approach, we integrate the microelectronic circuit and micro-Raman substrate into a microfluidic chamber and form a prototype of Raman biochip for biosensing. Enhancement of Raman signal and control of temperature of the sensor are both achieved. This technique provides a great potential for integrating microelectronics and micro-Raman sensors on a single glass chip.