This PDF file contains the front matter associated with SPIE proceedings volume 7553, including Title page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Optical resonances produced by photonic crystal surfaces can be designed to substantially amplify the electric field excitation of fluorophores while at the same time increasing the collection efficiency of emitted photons. Because PC surfaces can be produced inexpensively over large areas by replica molding, they offer an effective means for increasing sensitivity for broad classes of surface-based fluorescent assays. In this talk, the design and fabrication of photonic crystal surfaces and detection instrumentation for fluorescent enhancement will be described, along with demonstrated applications in gene expression microarrays and protein biomarker microarrays.

We have demonstrated real-time, label-free detection of small molecule binding using a novel optical biosensor. This sensor is a recently developed sensing platform incorporating a one-dimensional photonic crystal (PC) structure in a total-internal-reflection (TIR) geometry (PC-TIR). This simple configuration functions as an open Fabry-Perot resonator which provides a narrow optical resonance to enable label-free, highly sensitive detection of analyte molecules on the sensing surface in the enhanced evanescent field. Moreover, when the differential intensity modulation during binding is measured, a very high detection sensitivity can be obtained, and real-time binding observed. The well-studied biotinstreptavidin system was chosen to calibrate the detection limit for small molecule detection. Effective surface functionalization methods for streptavidin immobilization on the silica sensing surface were investigated, and analyte biotin molecules specifically binding to the sensing surface were monitored in real time. The binding of the smallest molecule D-Biotin, with a molecular weight of 244 Da, was easily experimentally observed with a high signal to noise ratio, which shows that the PC-TIR sensor has great potential to be a high-sensitivity and high-throughput sensing technology for small molecule binding analysis.

In this study, resonant microcavities in photonic crystal (PhC) waveguides are investigated for biosensing applications. The device architecture consists of a PhC waveguide with a defect line for guiding the transmission of light. Resonant microcavities created by changing the radius of a hole adjacent to the defect line are coupled to the PhC waveguide. Detection is based on shifts in the resonance wavelength observed in the transmission spectra. The PhC waveguide device is fabricated on silicon-on-insulator (SOI) wafers using electron beam lithography and reactive-ion etching (RIE). Receptor molecules are attached to the defects in the device by standard amino-silane and glutaraldehyde crosslinking chemistry. Preliminary results demonstrate successful detection of human IgG molecules as the target at large concentration levels of 500 μg/ml. Such PhC waveguide devices are advantageous for medical diagnostics and biosecurity applications as they allow rapid, label-free, and sensitive detection of multiple analytes in a single platform.

The rapid detection of foodborne pathogens is increasingly important due to the rising occurrence of contaminated food supplies. We have previously demonstrated the design of a hybrid optical device that has the capability to perform realtime surface plasmon resonance (SPR) and epi-fluorescence imaging. We now present the design of a microfluidic biochip consisting of a two-dimensional array of functionalized gold spots. The spots on the array have been functionalized with capture peptides that specifically bind E. coli O157:H7 or Salmonella enterica. This array is enclosed by a PDMS microfluidic flow cell. A magnetically pre-concentrated sample is injected into the biochip, and whole pathogens will bind to the capture array. The previously constructed optical device is being used to detect the presence and identity of captured pathogens using SPR imaging. This detection occurs in a label-free manner, and does not require the culture of bacterial samples. Molecular imaging can also be performed using the epi-fluorescence capabilities of the device to determine pathogen state, or to validate the identity of the captured pathogens using fluorescently labeled antibodies. We demonstrate the real-time screening of a sample for the presence of E. coli O157:H7 and Salmonella enterica. Additionally the mechanical properties of the microfluidic flow cell will be assessed. The effect of these properties on pathogen capture will be examined.

A rapid, automated, multi-analyte Microflow Cytometer is being developed as a portable, field-deployable sensor for onsite diagnosis of biothreat agent exposure and environmental monitoring. The technology relies on a unique method for ensheathing a sample stream in continuous flow past an interrogation region where optical fibers provide excitation and collect emission. This approach efficiently focuses particles in the interrogation region of the fluidic channel, avoids clogging and provides for subsequent separation of the core and sheath fluids in order to capture the target for confirmatory assays and recycling of the sheath fluid. Fluorescently coded microspheres provide the capability for highly multiplexed assays. Optical analysis at four different wavelengths identified six sets of the coded microspheres recognizing Escherichia coli, Listeria, and Salmonella as well as cholera toxin, staphylococcal enterotoxin B (SEB), and ricin, and assay results were compared with those of a commercial Luminex analysis system.

Magnetic modulation biosensing (MMB) system rapidly and homogeneously detected coding sequences of the nonstructural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA). A novel fluorescent resonance energy transfer (FRET)-based probe discriminated the target DNA from the control. When the target sequence is detected, the FRETbased probe is cleaved using Taq-polymerase activity and upon excitation with a laser beam fluorescent light is produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered into oscillatory motion by applying an alternating magnetic field gradient. The beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. Condensation of the beads from the entire volume increases the signal while modulation separates the signal from the background noise of the non-magnetized solution. 1.9 picomolar of the Ibaraki virus NS3 cDNA was detected in homogeneous solution within 18 minutes without separation or washing steps. In this paper we will review the magnetic modulation system and present its capability in specific DNA sequences detection.

The detection, identification and quantification of pathogenic microorganisms at low cost are of great interest to the agro-food industry. We have developed a simple, rapid, sensitive, and specific method for detection of food-borne pathogens based on use of nanoparticles alongside a low cost fluorescence cell reader for the bioassay. The nanoparticles are coupled with antibodies that allow specific recognition of the targeted Listeria in either a liquid or food matrix. The bioconjugated nanoparticles (FNP) contain thousands of dye molecules enabling significant amplification of the fluorescent signal emitted from each bacterium. The developed fluorescence Cell Reader is an LED-based reader coupled with suitable optics and a camera that acquires high resolution images. The dedicated algorithm allowed the counting of each individual nanoparticles-fluorescent bacterial cells thus enabling highly sensitive reading. The system allows, within 1 hour, the recovery and counting of 10⁴ to 10⁸ cfu/mL of Listeria in pure culture. However, neither the Cell Reader nor the algorithm can differentiate between the FNPs specifically-bound to the target and the residual unbound FNPs limiting sensitivity of the system. Since FNPs are too small to be washed in the bioassay, a dual tagging approach was implemented to allow online optical separation of the fluorescent background caused by free FNPs.

The ability to reproducibly and accurately control light matter interaction on the nanoscale is at the core of the field of optical biosensing enabled by the engineering of nanophotonic and nanoplasmonic structures. Efficient schemes for electromagnetic field localization and enhancement over precisely defined sub-wavelength spatial regions is essential to truly benefit from these emerging technologies. In particular, the engineering of deterministic media without translational invariance offers an almost unexplored potential for the manipulation of optical states with vastly tunable transport and localization properties over broadband frequency spectra. In this paper, we discuss deterministic aperiodic plasmonic and photonic nanostructures for optical biosensing applications based on fingerprinting Surface Enhanced Raman Scattering (SERS) in metal nanoparticle arrays and engineered light scattering from nanostructured dielectric surfaces with low refractive index (quartz).

Ultrathin porous silicon membranes provide a novel platform for label free detection and identification of biological samples using SERS. A 15 nm thin gold metal film was deposited on top of a 30 nm thick porous silicon membrane to form a thin porous metallic film. 3D FDTD simulations show EM field enhancement inside the holes together with increased scattering and extinction cross sections, making this structure a novel SERS substrate.

Analytical methods capable of in situ monitoring of water quality have been in high demand for environmental safety, the identification of minute impurities and fundamental understanding of potential risks of these molecular species. Raman spectroscopy, which provides 'fingerprint' information about molecular species in the excitation volume, is a powerful tool for in vivo diagnostics. However, due to a relatively weak Raman signal (~ 1 out of 10¹⁴ incident photons produces the useful signal) there is a need to significantly (by many orders of magnitude) enhance this signal, to raise the detection sensitivity of this technique. Traditionally, surface enhanced Raman spectroscopy is employed to dramatically increase the local field intensity and substantially improve the efficiency of Raman scattering. However, the above enhancement occurs only in "hot spots", which represent only a small percent of the total surface are of the substrate. Plasmonic nanostructures are also found to be hard to manufacture in large quantities with the desired degree of reproducibility and to be unable to handle high laser power. We propose and experimentally demonstrate a new type of approach for ultrasensitive Raman sensing. It is based on manufacturing a random porous structure of high-index material, such as GaP, and use the effect of light localization to help improving the detection sensitivity of such sensor. The desired structure was manufactured using electrochemical etching of GaP wafers. The observed Raman signal amplitudes are favorably compared to the best known plasmonic substrates.

Arrayed Imaging Reflectometry, or "AIR", is a new label-free optical technique for detecting proteins. AIR relies on binding-induced changes in the response of an antireflective coating on the surface of a silicon chip. Thus far, we have demonstrated the use of AIR for the detection of pathogenic E. coli, and for multiplex detection of a broad range of proteins in human serum. Creation of the near-perfect antireflective coating on the surface of silicon requires careful control over preparation of the chip surface prior to probe molecule immobilization. We present methods for highly reproducible, solution-phase silanization and glutaraldehyde functionalization of silicon chips carrying a layer of thermal oxide. Following functionalization with antibodies and passivation of remaining reactive groups, these surfaces provide exceptional performance in the AIR assay.

This work focuses on demonstrating proof-of-concept for a novel nanoparticle optical signal amplification scheme employing hybrid porous silicon (PSi) sensors. We are investigating the development of target responsive hydrogels integrated with PSi optical transducers. These hybrid-PSi sensors can be designed to provide a tunable material response to target concentration ranging from swelling to complete chain dissolution. The corresponding refractive index changes are significant and readily detected by the PSi transducer. However, to increase signal to noise, lower the limit of detection, and provide a visual read out capability, we are investigating the incorporation of high refractive index nanoparticles (NP) into the hydrogel for optical signal amplification. These NPs can be nonspecifically encapsulated, or functionalized with bioactive ligands to bind polymer chains or participate in cross linking. In this work, we demonstrate encapsulation of high refractive index QD nanoparticles into a 5wt% polyacrylamide hydrogel crosslinked with N,N'-methylenebisacrylamide (BIS) and N,N Bis-acryloyl cystamine (BAC). A QD loading (~0.29 wt%) produced a 2X larger optical shift compared to the control. Dissolution of disulphide crosslinks, using Tris[2-carboxyethyl] phosphine (TCEP) reducing agent, induced gel swelling and efficient QD release. We believe this hybrid sensor concept constitutes a versatile technology platform capable of detecting a wide range of bio/chemical targets provided target analogs can be linked to the polymer backbone and crosslinks can be achieved with target responsive multivalent receptors, such a antibodies. The optical signal amplification scheme will enable a lower limit of detection sensitivity not yet demonstrated with PSi technology and colorimetric readout visible to the naked eye.

Diffraction-based biosensors are often based on the adsorption of a target material on a grating made of thin layers, where the adsorption is detected by a modification of the diffracted signal. In this communication we discuss two strategies for enhancing this detection process. The first is based on the use of grating structures made of porous elements, where sensing is based on target molecules penetrating into the elements and modifying their effective index of refraction. The second is a resonant process where the effectiveness of the grating is enhanced by the coupling to surface electromagnetic states, in particular Bloch surface waves that exist at the interface between a homogeneous medium and a photonic crystal.

In this work, we theoretically and experimentally demonstrate a highly sensitive polymer-cladded porous silicon (PSi) membrane waveguide based on a ~1.55 μm thick porous silicon membrane coated on one side with a low loss polymer. The sensor operates in the Kretschmann configuration, which is amenable to microfluidics integration, with a high index cubic zirconium prism. The sensitivity of the sensor is investigated through PNA hybridization in the PSi membrane. We demonstrate that higher angle resonances and a proper ratio of PNA length to PSi pore diameter lead to significantly improved detection sensitivity. A detection sensitivity below 0.1°/μM is reported for 16mer target PNA. Calculations and complimentary experiments show that careful tuning of the polymer cladding thickness can further improve the detection performance.

Label-free optical biosensors based on photonic crystal slabs offer high sensitivity and simplified coupling to incoming free-space radiation. Biosensing results from detecting shifts in guided resonance frequency spectral location based on changes in index of refraction due to analytes binding at the PC slab surface. We have evaluated biosensing for a PC slab suspended above a substrate, and compared it to a case where the PC slab lies directly on a substrate. Differences in guided resonance quality factors (Q) were largely invariable for slab-on-substrate and suspended PC slab designs. However, we show that index of refraction sensitivity in a suspended PC slab can be enhanced nearly three-fold over the slab-on-substrate design.