This PDF file contains the front matter associated with SPIE Proceedings Volume 6759, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Microfluidics offer the advantages of multiplexed analysis on small, inexpensive platforms. We describe herein two distinct optical detection techniques that have the common point of sequestering and measuring analyte signals in highly localized EM fields. The first technique mates a microfluidic polydimethylsiloxane (PDMS) platform with colloidal-based surface enhanced Raman scattering (SERS) in order to perform parallel, high throughput vibrational spectroscopy. Spectra are acquired for analytes localized in surface plasmon fields associated with conventional and uniquely synthesized cubic silver colloids. SERS studies such as pH of the colloidal solution, and the type of colloid are used to demonstrate the efficiency and applicability of the method. In addition, a facile passive pumping method is used to deliver Ag colloids and analytes into the channels where all SERS measurements were completed under nondestructive flowing conditions. With this approach, SERS signal reproducibility was found to be better than 7%. A calibration curve for the drug mitoxantrone (resonance enhanced) was generated. The second technique seeks to integrate a passively-pumped, microfluidic, PDMS platform and planar waveguide technology, utilizing magnetic beads as solid supports for fluoro-assays with direct detection of bound analyte within the sample mixture accomplished by selectively driving functionalized beads to a localized evanescent field. Because analyte binding occurs in free solution, the reaction is not diffusion limited and, once magnetically delivered to the evanescent wave, the analyte can be detected with fewer complications arising from non-optically homogeneous, biological matrices. Additionally, the evanescent sensing surface can be easily regenerated by simply removing the bead-retaining magnetic field. Initial testing, optimization and calibration were performed using a model sandwich immunoassay system for the detection of rabbit IgG, with which we demonstrate a linear dynamic range of 3 orders of magnitude and physiologically relevant detection limits of nanograms per milliliter.

Surface enhanced Raman spectroscopy (SERS) utilizing silver colloids for localized plasmonic enhancement has been heavily researched due to its tremendous increase in the Raman signal of bio/chemical molecules. We demonstrate further enhancement by multiplying the SERS effect by the resonant enhancement of a ring resonator microcavity. The liquid core optical ring resonator (LCORR) offers a high-performance and practical design to obtain this composite enhancement for bio/chemical molecule detection. The LCORR integrates an array of optical ring resonators into a capillary-based microfluidic channel to form a novel bio/chemical sensing platform. The circular cross-section of the glass capillary acts as an optical ring resonator, with the evanescent field of the resonant light interacting with the sample passing through the capillary. The LCORR has already been well-studied for applications in label free biomolecule sensing. In this work, we utilize a silver colloid solution inside the capillary to perform SERS-based detection. In contrast to a typical SERS system where the incident light interacts with the colloid and target molecules only once, in the LCORR system, the tightly confined light resonates around the capillary wall, repeatedly interacting with the SERS system. Our experimental results show the increased enhancement due to the composite effect of the cavity resonance and the localized plasmonic effect of the nanoparticles inside the cavity. We have achieved detection of 3.3 nM R6G inside the LCORR. In addition to the excellent sensitivity, this detection system represents an advancement in the development of practical SERS bio/chemical sensors due to the arrayed nature of the sensors combined with the integrated microfluidics of the LCORR.

A hyperspectral Fourier transform spectrometer (HS-FTS) has been developed to study biological material binding to surfaces through spatially resolved, spectral self-interference fluorescence microscopy and also label-free white light reflectance spectroscopy. Spectral self-interference fluorescence microscopy yields the height of fluorescent tags bound to a specific location on biomolecules tethered to a surface, and from this the biomolecule conformation can be predicted; white light reflectance spectroscopy yields the average height of an ensemble of biomolecules relative to the surface. The HS-FTS is composed of a small, step scanning Michelson interferometer made by Optra, Inc., a series of commercial off the shelf imaging lenses, and a 12-bit thermoelectrically-cooled CCD camera. The system operates over the 500 to 900 nm spectral range with user defined spectral resolution, thereby supporting use of a host of fluorescent tags or white light spectral windows. The system also supports near real-time hyperspectral cube acquisition via undersampling with the use of a spectral filter and user defined interferometer step increments. The overall approach offers flexible yet sensitive measurement capability for a variety of biological studies. Preliminary results are presented of both spectral self-interference fluorescence microscopy and white light reflectance spectroscopy measurements of artificial, photographically etched surfaces with feature heights on the order of 10 nm. Planned future work includes spectral self-interference fluorescence microscopy measurements of biomolecule conformation as manipulated by external electrical and magnetic fields as well as label-free white light reflectance spectroscopy measurements of DNA microarrays.

The proposed micro-spectrometer consists of multi-slit grating and bolometric IR detector array. The grating is used for dispersion of an incident optical radiation into different wavelength components. The grating pattern is made by metal RIE of aluminum deposited on dielectric membrane, which is fabricated over silicon wafer. The IR detector part adopts a resistive bolometer that changes its resistance when a temperature change occurs. Due to the temperature rise by an incident IR ray, the presence of IR can be detected by knowing the resistance change on the bolometer. For the material of resistive bolometer, vanadium oxide is utilized. Vanadium oxide is adopted for resistive bolometer in this work because of its high TCR.

This paper presents a novel miniature sensing tip structure for various biomedical applications. With such a tiny tip, the sensor has potential to be inserted into cells for intracellular measurements without any label as indicator. This label-free detection method is very useful in biological areas such as DNA hybridization detection and antigen-antibody interaction monitoring. Single-cell analysis (SCA) technology can provide dynamic analysis of interactions within individual living cells, in addition to providing a complement to conventional bulk cell assays. When the number of sample cells obtained from surgical procedures is limited, and cannot be propagated for study, SCA is especially important. It provides a valuable tool for intracellular studies that have applications ranging from medicine to national security. In addition, the sensor fabrication is simple and has potential for batch manufacturing. The sensor performance will be reproducible and uniform. Uniformity and reproducibility are two very important requirements for sensor manufacturing. Unfortunately, most current optical fiber sensors are hand-made one by one, and the sensors' performance is not easy to be uniform. Our novel sensor will be able to address this problem. This may lead to batch processing and a great reduction of the fabrication cost.

Across extracellular surfaces, lipid rafts are believed to be an important organizing membrane microdomain component, facilitating specific protein-protein interactions by selectively excluding or including proteins into them. The lipid-based sorting mechanism of these microdomains has been implicated in many cellular processes including; membrane trafficking, signal transduction and cell growth regulation. However, since individual rafts are estimated to range in size from the nanoscale to the microscale, in many cases, they cannot be easily monitored by conventional imaging techniques. We have developed surface enhanced Raman scattering (SERS)-based nanoimaging probes for nanoscale imaging of biochemical species on and within extracellular environments. These probes synergistically combine the qualitative and quantitative information of SERS with the nanoscale imaging capabilities of tapered fiber optic bundles, potentially allowing for chemical imaging of extracellular components and chemical exchange events across cellular surfaces. These probes are fabricated from coherent fiber optic bundles containing 30,000 individual fiber elements that have been tapered to have diameters as small as 140 nm, thus allowing for image magnification and submicron spatial resolution. Due to the uniformly roughened surface features across the probe's imaging surface onto which silver island arrays are fabricated, these probes exhibit less than 3% RSD in SERS signal across the imaging area. In this work, tunability, multiplex detection capabilities and an application of these SERS nanoimaging probes to biological systems are demonstrated.

Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

Fiber optic based sensor technologies have many significant advantages over electrochemical sensors, and as a result have broad application for sensing in biology, agriculture and medicine. An important component of fiber optic biosensor is the sensing element. Usually, a polymer matrix containing the analyte specific fluorescent dye is immobilized on one end of the fiber optic probe. The polymer matrix provides mechanical stability to the immobilized membrane and entraps the fluorescent dye molecules. The target analyte diffuses into the membrane polymer and quenches the fluorescent dye. This optical interaction between the analyte and fluorescent dye dynamically changes the fluorescence lifetime of the dye. These changing fluorescent lifetimes reveal information about the target analyte concentration. Although the fluorescent lifetime of the dye is of primary interest, high signal-to-noise ratio (SNR) is also very desirable. Conventionally, complex electronics is implemented to achieve high SNR. Various signal processing methods such as signal amplification and filtering are implemented to achieve high SNR. In this study, we report optical signal amplification by modification of the dye-polymer matrix by addition of titanium oxide particles. The addition of titanium oxide particles enhanced the optical signal intensity. Optical performances of different sizes and concentrations of titanium oxide particles are compared. We believe that this increased optical intensity is due to increased optical scattering in the dye-polymer matrix. We also compare the performance of titanium oxide particles to gold and other material particles to experimentally probe the exact nature of light scattering in the dye-polymer matrix.

We have been investigating a new nanomaterial-based optical platform for the immobilization of protease substrates for the development of a biosensor to detect medically relevant enzymes. Metallic nanoparticles have been deposited onto thin films and are being used for their optical properties. Two different peptide sequences have been designed as trypsin substrates that are designed to be immobilized onto the surface of the thin films. The peptides were synthesized with a fluorophore attached at the terminal end of the peptide to allow for fluorescence sensing. Fluorescent molecules in close proximity to metallic elements will have their fluorescence signal quenched due to surface plasmon resonance (SPR) effects. When the peptide is cleaved by trypsin, the fluorophore is separated, resulting in a detectable change in fluorescence intensity. These novel nanomaterial-based optical platforms have been fabricated using physical vapor deposition. Innovative techniques have been invented using these machines to acquire nanoparticles in the range of a few nanometers on these thin films. It is known that nanoparticles with dimensions less than their bandwidth display optical properties much different from their bulk counterparts. We have immobilized the peptide substrates to the surface of the metallized thin films so they are in close proximity with each other. Polydimethlysiloxane (PDMS) was molded to create small wells and placed on the thin films. Fluorescent microscopy was used to image the wells as various concentrations of the enzyme were introduced resulting in a recovery of green fluorescence from the fluorophore on the cleaved portion of the peptide. Different size nanoparticles and different immobilization processes are being used to optimize the design of the protease biosensor.

Gold-based surface-enhanced Raman scattering (SERS) beacons have been developed, which represent a simple, biocompatible and rapid means of performing multiplexed DNA sequence detection in a non-arrayed format. These SERS beacons consist of a simple stem-loop oligonucleotide probe in its native form with one end attached to a SERS active dye molecule and the other to a gold nanoparticle, approximately 50 nm in diameter. The probe sequence is designed to achieve a stem-loop structure, with the loop portion complementary to the target sequence, similar to fluorescent molecular beacons. In the absence of the target DNA sequence, the SERS signal of the associated dye molecule is detected, representing the "ON" state of the probe. When the target sequence is hybridized to the probe, which results in an open conformation, its respective reporter dye is separated from the gold nanoparticle, producing diminished SERS signal. In this paper, the fabrication and characterization of these SERS beacons is described. We also demonstrate selective hybridization of a target sequence to one beacon in a mixture, revealing their potential for use in a multiplexed fashion.

A new temperature sensing technique based on high-resolution reflection measurement in a fiber optic system is demonstrated. The technique adopts thermoreversible compounds as a transducer in a fiber-optic temperature sensor system. The dependence of the light reflection on the temperature has been studied. The reflected optical power indicated a change in the temperature range of observation, corresponding to a change of 15.3dB in the reflection signal when the temperature increased from 20°C to 140°C. The temperature dependence of the optical properties was found to be reversible, which indicates that the temperature transducer is reversible as well.

Glucose binding protein (GBP) is a monomeric periplasmic protein. It is synthesized in the cytoplasm of Escherichia coli which functions as a receptor for transport D-glucose. GBP binds glucose with high affinity. The binding mechanism is based on a hinge motion due to the protein conformational change. This change was utilized as an optical sensing mechanism by applying Fluorescence Resonance Energy Transfer (FRET). The wild-type GBP lacks cysteine in its structure, but by introducing a single cysteine at a specific site by site-directed mutagenesis, this ensured single-label attachment at specific sites with a fluorescent probe. The other sites were amino sites, which were labeled with second fluorophore. The near IR FRET pair, Alexa Fluor 680 (AF680) and Alexa Fluor 750(AF750), was utilized. The AF680 targeted the amine sites, which was the donor fluorophore, while the AF750 labeled the single cysteine site, which was the acceptor fluorophore. The sensing system strategy was based on the fluorescence changes of the probe as the protein undergoes a structural change upon binding. This biosensor had the ability to detect down to 10 uM concentrations of glucose. Next the probes were uploaded into red blood cells via hypo osmotic dialysis. The sensor responded to glucose while encapsulated with the red cells. These results showed the feasibility of an intracellular glucose biosensor.

Laser induced photothermal radiometry (PTR) was applied as a safe, non-destructive, and highly sensitive tool for the detection of early tooth surface demineralization. In the experiments, teeth were treated sequentially with an artificial demineralization gel to simulate controlled mineral loss on the enamel surface. Modulated laser light generated infrared blackbody radiation from teeth upon absorption and nonradiative energy conversion. The infrared flux emitted by the treated region of the tooth surface and sub-surface was monitored with an infrared detector twice: before and after treatment. The experiments showed very high sensitivity of the measured signal to incipient changes in the enamel structure, emphasizing the clinical capabilities of the method. In order to analyze the biothermophotonic phenomena in a sample during the photothermal excitation, a theoretical model featuring coupled diffuse-photon-density-wave and thermal-wave fields was developed. The theoretical fits based on the three-layer approach (demineralizad enamel + healthy enamel + dentin) allowed fitting thermal and optical properties of the demineralized layer. The theoretical analysis showed that the dentin layer should be taken into account in the fittings.

Multiphoton excitation of exogenous dyes and endogenous biochemical species has been used extensively for tissue diagnosis by fluorescence spectroscopy. Unfortunately, the majority of endogenous biochemical chromophores have low quantum yields, less than 0.2, therefore determining two-photon cross sections of weakly luminescencing molecules is difficult using two-photon fluorescence spectroscopy. Accurate determination of two-photon cross sections of these biochemicals could provide insight into fluorescence signal reduction caused by the absorption of excitation energy by non-target intracellular species. Non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) is a novel technique we have developed for condensed matter measurements that has the potential for accurately determining two-photon absorption cross-sections of chemicals with small or non-existant fluorescence quantum yields. In this technique, near infrared light is used to generate an ultrasonic signal following a non-resonant two-photon excitation process. This ultrasonic wave is directly related to the non-radative relaxation of the chromophore of interest and is measured using a contact piezoelectric ultrasonic transducer. The signal from the ultrasonic transducer can then be used to calculate two-photon absorption cross sections. This paper will describe the validation of this technique by measuring the two-photon absorption cross- sections of well characterized chromophores such as rhodamine B and coumarin 1 in solution as well as riboflavin in a gelatin tissue phantom.

Directional diffusivities derived from diffusion tensor magnetic resonance imaging (DTI) measurements describe water movement parallel to (λ_||, axial diffusivity) and perpendicular to (λ⊥radial diffusivity) axonal tracts. λ_|| and λ⊥ have been shown to differentially detect axon and myelin abnormalities in several mouse models of central nervous system white matter pathology in our laboratory. These models include experimental autoimmune encephalomyelitis (EAE), (1) myelin basic protein mutant mice with dysmyelination and intact axons, (2) cuprizone-induced demyelination, and remyelination, with reversible axon injury (2, 3) and a model of retinal ischemia in which retinal ganglion cell death is followed by Wallerian degeneration of optic nerve, with axonal injury preceding demyelination. (4) Decreased λ|| correlates with acute axonal injury and increased λ⊥ indicates myelin damage. (4) More recently, we have translated this approach to human MR, investigating acute and chronic optic neuritis in adults with multiple sclerosis, brain lesions in adults with multiple sclerosis, and acute disseminated encephalomyelitis (ADEM) in children. We are also investigating the use of this technique to probe the underlying structural change of the cervical spinal cord in acute and chronic T2- hyperintense lesions in spinal stenosis, trauma, and transverse myelitis. In each of these demyelinating diseases, the discrimination between axonal and myelin injury which we can achieve has important prognostic and therapeutic implications. For those patients with myelin injury but intact axons, early, directed drug therapy has the potential to prevent progression to axonal loss and permanent disability.

Underground miners are exposed to some of the highest levels of diesel particulate matter (DPM) in the United States. Therefore, it is important to monitor the exposure of miners to DPM, but it can be difficult because of the complex composition of DPM and the number of interferences. Currently, elemental carbon (EC) is used as a surrogate because it makes up a significant fraction of the DPM and is not affected by interferences. Standard measurement methods for EC can be time consuming and only record end of shift results. In this research, a laser absorption technique that enables one to measure EC concentration in near real time was shown to be a beneficial tool. The real time data showed that the fresh air being drawn into a stone mine was not properly reaching the working area and needed to be redirected to decrease DPM concentrations. The real time data also provided a more accurate efficiency of an environmental cab compared to just using the standard method by detecting the opening of the cab's window and door. The EC optical monitor was also worn by researchers in a mine to show how it can give not only the average concentration for the shift but also reveal when and where a miner is exposed to DPM.

When a finger is pressed against a flat plate and deformed, blood inside the finger moves away from the deformed area. This causes the finger to change its appearance from reddish to white. As the finger leaves the plate, the blood comes back and it looks reddish again. We have proposed to use this color change to distinguish genuine fingers from artificial ones for un-attended fingerprint identification systems. This blood-related signal may reflect the stiffness of the peripheral blood vessels and therefore it may be correlated with some health conditions such as blood pressure. In experiments, we used a fingerprint sensor based on scattered light detection. Because the spectra of the light scattered by the deformed fingers showed large changes mostly in the green portion, an LED emitting at 525 nm at peak strength was used. First, we compared series of fingerprint images acquired during a normal input action and those obtained while a rubber band occluded the blood flow. The occluded finger required a larger force to exhibit a similar change for these pixel values than the finger without the rubber band. Second, we analyzed fingerprint images recorded by six volunteers. We defined some indices based on the pixel values of the fingerprint images and the pressure applied to the fingers. The correlation coefficient of one of such indices and the average blood pressure of the participants was 0.86. Although the number of the subjects is small, this initial result is encouraging.

Nucleic acid aptamers can exhibit high binding affinities for a wide variety of targets and have received much attention as molecular recognition elements for enhanced biosensor performance. These aptamers recognize target molecules through a combination of conformational dependent non-covalent interactions in aqueous media which can be investigated using capillary electrophoresis-based methods. In this paper we report on the results of our studies of the relative binding affinity of Campylobacter jejuni aptamers using a capillary electrophoretic immunoassay. Our results show preferential binding to C. jejuni over other common food pathogen bacteria. Capillary electrophoresis can also be used to develop new aptamer recognition elements using an in vitro selection process known as systematic evolution of ligand by exponential enrichment (SELEX). Recently, this process has been adapted to use capillary electrophoresis in an attempt to shorten the overall selection process. This smart selection of nucleic acid aptamers from a large diversity of a combinatorial DNA library is under optimization for the development of aptamers which bind to Army-relevant targets. This paper will include a discussion of the establishment of CE-SELEX methods for the future development of smart aptamer probes.

We present an intelligent microfluidic system with oxidase enzyme coupled biosensors. Baseline (zero-value) drift and sensitivity degradation are two common problems related with biosensors. In order to overcome these problems there is a great need for integrating an on-demand, in situ self-diagnosis and self-calibration unit along with the sensor. Utilizing the microfluidic technology, we explore the feasibility of implementing this function without any externally coupled bulky apparatus. A microsystem including a microfluidic channel and calibration electrodes are prepared by microfabrication techniques. A novel method of using hydrogen and oxygen bubbles generated by electrolysis of water is used to saturate the solution with these gases in the microfluidic channel where the biosensor is placed. The hydrogen bubble provides oxygen-depleted microenvironment to conduct a zero-value calibration procedure for the sensor. The oxygen bubble provides high sensitivity and constant oxygen background environment to allow stable enzyme reactions that is not limited or perturbed by the fluctuation of background oxygen in sample solutions. Commercial oxygen sensors and pH sensors are used to confirm whether saturation or depletion of oxygen has occurred with minimum local pH change near the sensor during the electrolytic bubble generation. The glucose data obtained from the experiments assure that our proposed method is promising to overcome the above mentioned two problems.