The rapid and accurate determination of bacterial spores in sterile environments (i.e., food preparation areas and hospitals) is of great importance in the minimization of bacteria-driven illnesses. Surface- Enhanced-Raman-Scattering (SERS) is potentially a very sensitive spectroscopic technique for the detection of biological threat agents. SERS can be made a more robust and attractive spectroscopic technique for biological threat agent sensing by developing high sensitivity, high reproducibility SERS-active substrates. The maturation of Si-based semiconductor fabrication techniques has facilitated the development and marketing of highly reproducible and sensitive SERS-active substrates. Klarite^TM SERS-active substrates (Mesophotonics LTD) have been analyzed for utility in the potential identification of biological threat agents. Concurrently, we have investigated prototype SERS- active substrates under development by Nanospectra Biosciences, Inc. Substrate spatial reproducibility and substrate-to-substrate reproducibility will be discussed.

We have developed a novel class of surface-enhanced Raman scattering (SERS)-based nanoimaging probes for chemical imaging with nanometer scale spatial resolution. Using these SERS nanoimaging probes it is possible to differentiate between different chemical components within the sample under analysis. These SERS nanoimaging probes are fabricated from coherent fiber optic imaging bundles composed of 30,000 individual image transmission elements. Using a specially programmed micropipette puller, the individual fiber elements are uniformly heated and pulled, creating a tapered bundle with a flat tip. In order to create the SERS active surface, the tapered end of the fiber bundle is roughened on the "molecular scale" via chemical etching, and is then over-coated with silver in a controlled manner via vapor deposition in a vacuum evaporator. Following etching, six regularly spaced peaks are produced surrounding each of the individual image transmission elements of the bundle, and it is these peaks onto which the metal over-layer is deposited. Due to the high degree of uniformity in the surface of these tapered and etched tips, these SERS nanoimaging probes exhibit significantly greater reproducibility than SERS substrates fabricated through similar silver film over nanostructure fabrication processes. Characterization and optimization of these SERS nanoimaging probes using SERS active chemicals is discussed.

Three-dimensional gold nanowire ensembles (NEE) are a novel and useful platform for electrochemical DNA detection. Work performed in our laboratory using the three-dimensional nanostructures with an electrocatalytic reporter system has produced attomole sensitivity towards target DNA sequences. Large electrocatalytic signals observed at DNA-modified nanowires produce high signal-to-noise ratios, which is one factor that contributes to the improved sensitivity. DNA- modified nanostructures generate amplified electrocatalysis signals that are significantly larger than those observed at bulk gold surfaces, and our experiments indicate that the three-dimensional architectures of the nanowires facilitate the electrocatalytic reaction because of enhanced diffusion and accessibility occurring around these structures. The heightened sensitivity achieved indicates that the nanowire ensembles constitute a promising platform for ultrasensitive biosensors.

The combination of electrochemistry with microfluidic sample processing is a viable option to reduce the size, logistics load and power consumption of biosensors. Modern microfluidics technology makes it possible to perform sample clean-up, PCR, sample concentration and transduction on the same disposable chip. This presentation will discuss two novel electrochemical techniques which do not require a sandwich assay and can be employed on a disposable microfluidic chip, reducing logistics load and microfluidic complexity. Transduction is achieved via an electrochemical DNA hybridization sensor similar to a molecular beacon removing the need for a sandwich assay also referred to as E-DNA. The sensor is designed where a DNA stem-loop structure is immobilized on a gold electrode with a redox label held close to the surface. Upon hybridization the stem-loop opens and the label pulls away from the surface so that current cannot flow to the electrode under positive bias. This paper will primarily discuss experiments trying to understand the hybridization event and effect of surface morphology on electrochemical signal transduction.

Multiple layers were attached to the gold surface of surface plasmon resonance (SPR) sensor to maximize the antibody loading and the specific signal of an antigen, while minimizing non-specific signal from full serum proteins. A three-fold improvement of the specific signal from myoglobin and a three-fold decrease of non-specific signal from serum were observed using the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) compared to the currently commercially available carboxymethylated dextran. Self-assembled monolayers were attached to the gold surface. 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4,4'-dithiodibutyric acid, 11- mercaptoundecanoic acid, and 16-mercaptohexadecanoic acid were investigated. The covalent attachment of the layers was monitored using SPR and GATR-FTIR. Antibodies to human myoglobin were covalently attached to the sensor using EDC / NHS chemistry and detection of 25 ng/mL myoglobin solution was monitored for the specific signal. Exposing the sensors with the layers to full bovine serum, protein concentration of 72 mg/mL, monitored non-specific signal. NHS-MHA was used to quantify proteins cell culture media with limits of detection below 1 ng/mL.

Salicylidene derivatives have been developed as a new set of highly sensitive chromogenic and fluorogenic sensors for anions. A systematic synthesis and investigation of salicylidene derivatives reveals their complementarity with fluoride, acetate and phosphate ions, which can be utilized in anion-sensor binding. Synthesis of receptors proceeded at room temperature or by microwave irradiation, both achieving yields of 80-95 percent with a short reaction time. A substantially red-shifted fluorescence and absorption of receptors in acetonitrile was enhanced upon addition of fluoride, acetate and dihydrogen phosphate depending on the extent of conjugation, nature and the site of substituents in the system. Intense color change and high binding constants demonstrates high sensitivity for fluoride ions as compared to other anions that could allow detection under both visual and fluorescence emission conditions. Investigation of behavior of receptors with anions in different solvents reveals the interaction to be hydrogen bonding in nature.

Monitoring the presence, production and transport of proteins inside individual living cells can provide vital information about cellular signaling pathways and the overall biological response of an organism. For example, cellular response to external stimuli, such as biological warfare (BW) agents, can be monitored by measuring interleukin-II (IL-2) expression inside T-cells as well as other chemical species associated with T-cell activation. By monitoring such species, pre-symptomatic detection of exposure to BW agents can be achieved, leading to significantly increased post-exposure survival rates. To accomplish such monitoring, we have developed and optimized implantable nanosphere-based nanosensors for the intracellular analysis of specific proteins in a label-free fashion. These sensors consist of 300-520 nm diameter silica spheres that have been coated with silver and antibodies to allow for trace protein detection via surface enhanced Raman spectroscopy (SERS). They have been optimized for SERS response by evaluating the size of the nanospheres best suited to 632.8 nm laser excitation, as well as the various nanosensor fabrication steps (i.e., silver deposition process, antibody binding, etc.). During usage, the presence of the specific protein of interest is monitored by either directly measuring SERS signals associated with the protein and/or changes in the SERS spectrum of the antibodies resulting from conformational changes after antigen binding. In this work, human insulin was used as a model compound for initial studies into the sensitivity of these optimized nanosensors.

Raman spectroscopy is a powerful technique for rapid, non-invasive and reagentless analysis of materials, including biological cells. In many samples of biological origin, laser illumination leads to luminescence in addition to Raman scattering. This luminescence will often dissipate after prolonged laser exposure. A common practice is to allow a sample to "photobleach" prior to acquisition of a high quality Raman spectrum. In an effort to automate data acquisition on such samples we are investigating an automated means of quantifying photobleaching and acquiring Raman spectra after photobleaching. We present results of a comparison between an automated approach to acquiring a spectrum on a sample with dynamic luminescence and a manual approach taken by a trained spectroscopist. This component of smart biomedical sensor technology will help allow high quality spectral data to be acquired reproducibly to potentially aid non-spectroscopists with application of Raman spectroscopic approaches.

A design for a high-resolution scanning instrument is presented for in vivo imaging of the human eye at the cellular scale. This system combines adaptive optics technology with a scanning laser ophthalmoscope (SLO) to image structures with high lateral (~2 μm) resolution. In this system, the ocular wavefront aberrations that reduce the resolution of conventional SLOs are detected by a Hartmann-Shack wavefront sensor, and compensated with two deformable mirrors in a closed-loop for dynamic correction and feedback control. A laser beam is scanned across the retina and the reflected light is captured by a photodiode, yielding a two-dimensional image of the retina at any depth. The quantity of back-scattered light from the retina is small (0.001% of reflection) and requires the elimination of all parasite reflections. As an in vivo measurement, faint cellular reflections must be detected with a low-energy source, a supraluminescent laser diode, and with brief exposures to avoid artifacts from eye movements. The current design attempts to optimize trade-offs between improved wavefront measurement and compensation of the optical aberrations by fractioning the light coming to the wavefront sensor, better sensitivity by increasing the input light energy or the exposure time and the response speed of the system. This instrument design is expected to provide sufficient resolution for visualizing photoreceptors and ganglion cells, and therefore, may be useful in diagnosing and monitoring the progression of retinal pathologies such as glaucoma or aged-related macular degeneration.

Optical Coherence Tomography (OCT) was recently proposed by our group for noninvasive, continuous monitoring of blood glucose concentration in diabetics as well as in critically ill patients (both diabetic and nondiabetic). In our previous studies we tested OCT-based glucose sensing using one-dimensional (1-D) lateral scanning of the OCT probing beam over the tissue surface. The measured OCT signal was prone to motion artifacts and had high level of speckle noise. In this study we used two-dimensional (2-D) lateral scanning of the OCT probing beam and achieved 3.6-fold reduction of the speckle noise level. We also applied a post-processing Fourier filtration technique that resulted in an additional 2-2.5-fold suppression of noise. Our data indicate that the combination of acquisition time of 30-40s and the Fourier filtration technique may provide OCT monitoring of blood glucose concentration with a sensitivity of 1mM (18 mg/dL).

Ultra-wideband radar holds great promise for a variety of medical applications. We have demonstrated the feasibility of using ultra-wideband sensors for detection of internal injuries, monitoring of respiratory and cardiac functions, and continuous non-contact imaging of the human body. Sensors are low-power, portable, and do not require physical contact with the patient. They are ideal for use by emergency responders to make rapid diagnosis and triage decisions. In the hospital, vital signs monitoring and imaging application could improve patient outcomes. In this paper we present an overview of ultra-wideband radar technology, discuss key design tradeoffs, and give examples of ongoing research in applying ultra-wideband technology to the medical field.

In order to measure muscle physiological parameters such as pH and oxygen partial pressure (PO2) by continuous wave (CW) diffuse reflectance near-infrared spectroscopy (NIRS), light must penetrate through skin and subcutaneous fat layers overlying muscle. In this study, the effect of skin and subcutaneous fat layer and on the spatial sensitivity profile of CW diffuse reflectance near-infrared spectra is investigated through Monte Carlo simulations. The simulation model uses a semi-infinite medium consisting of skin, fat and muscle. The optical properties of each layer are taken from the reported optical data at 750 nm. The skin color is either Caucasian or Negroid and the fat thickness is varied from 0 ~ 20 mm. The spatial sensitivity profile, penetration depth, and sensitivity ratio as functions of optical fiber source-detector separation (SD, 2.5 mm, 5.0 mm, 10.0 mm, 20.0 mm, 30.0 mm and 40.0 mm), skin color and fat thicknesses are predicted by the simulations. It is shown that skin color only slightly influenced the spatial sensitivity profile, while the presence of the fat layer greatly decreased the detector sensitivity. It is also shown that probes with longer SD separations can detect light from deeper inside the medium. The simulation results are used to design a fiber optic probe which ensures that enough light is propagated inside the muscle in NIRS measurement on a leg with a fat layer of normal thickness.

Muscle pH is an important indicator of inadequate blood flow and available oxygen. Muscle pH can be used to triage and help treat trauma victims and indicate poor peripheral blood flow in diabetic patients. Muscle pH can also be used to indicate exercise intensity and fatigue. We have developed methods to non-invasively measure muscle pH using Near-Infrared Spectroscopy (NIRS) and Partial Least Squares (PLS) analysis. A multi-subject PLS model correlating near infrared tissue spectra, acquired from healthy subjects during repetitive hand-grip exercise, to invasive tissue pH measurements, has been developed and validated. Subject related variations in the spectral signal; impede the development of viable multi-subject model. Within-subject variations in tissue NIR spectra often result from uncontrolled motion or blood volume changes during exercise, while subject-to-subject variations arise from differences in skin pigmentation and the fat layer thickness. We have developed signal processing techniques to account for these mitigating factors. By incorporating this signal processing techniques with PLS calibration, we can generate a pH model that has a relative standard error of prediction of 1.7%

The application of multivariate calibration models, specifically those using partial least squares (PLS) regression to relate near infrared (NIR) spectral data to analyte concentrations, relies upon accurate knowledge of the concentrations during model building. In a physiologic system, such as human skeletal muscle, these concentrations can be measured using invasive sensors which may have material properties that limit diffusion of analytes to the sensing chemistry, thus taking several minutes to fully respond to an analyte change which actually occurs in seconds. This results in a poor time correlation between reference measurements of analyte concentrations and spectral data, which in turn degrades the performance of the PLS model. We mathematically modeled the response of an invasive sensor measurement and used this response to develop a filter to time-match the raw NIR spectra before building the PLS model. PLS models for interstitial pH in exercising human flexor digitorum profundus muscle were developed with and without the time-matching filter. In a single exercising subject, root mean square error of prediction (RMSEP) = 0.05 pH units and r² = 0.39 without filtering, but improved to RMSEP = 0.02 pH units with r² = 0.91 after the time-matching filter was implemented. The time-matching filter was shown to be effective in improving model performance when spectral response is more rapid than the invasive sensor reference measurement.

Brain cancer affects approximately 16,500 people a year and individuals diagnosed with glioblastoma multiforme have an average life expectancy of less than 12-18 months after diagnosis. A portable fiber-optic probe capable of distinguishing between healthy and tumor tissues, with a high degree of spatial resolution, deep within a sample would be a valuable tool for tumor diagnosis and margining. A novel technique that combines 1-2 cm penetration depths with cellular level spatial resolution to chemically distinguish cancerous from non-cancerous tissues is non-resonant multiphoton photoacoustic spectroscopy (NMPPAS). This technique focuses pulsed near infrared light into a sample, creating a two-photon excitation event, and measures the resulting non-radiative decay as an ultrasonic signal. This paper discusses the optimization of a portable fiber-optic NMPPAS probe capable of delivering nanosecond laser pulses from 740nm-1100nm to a series of lens, which focus the light into the sample. The resulting ultrasonic signal is measured using a polyvinylidene fluoride based piezoelectric detector. The two-photon excitation efficiency of the portable NMPPAS probe system has been evaluated by measuring the two-photon excitation and emission spectra of common fluorescent dyes such as rhodamine B and fluorescein. In addition, this paper also demonstrates the diagnostic potential of this technique for tumor detection and margining without the need for acquisition of an entire spectrum.

The purpose of this study was to evaluate the performance of a bimorph deformable mirror from AOptix, inserted into an adaptive optics system designed for in-vivo retinal imaging at high resolution. We wanted to determine its suitability as a wavefront corrector for vision science and ophthalmological instrumentation. We presented results obtained in a closed-loop system, and compared them with previous open-loop performance measurements. Our goal was to obtain precise wavefront reconstruction with rapid convergence of the control algorithm. The quality of the reconstruction was expressed in terms of root-mean-squared wavefront residual error (RMS), and number of frames required to perform compensation. Our instrument used a Hartmann-Shack sensor for the wavefront measurements. We also determined the precision and ability of the deformable mirror to compensate the most common types of aberrations present in the human eye (defocus, cylinder, astigmatism and coma), and the quality of its correction, in terms of maximum amplitude of the corrected wavefront. In addition to wavefront correction, we had also used the closed-loop system to generate an arbitrary aberration pattern by entering the desired Hartmann-Shack centroid locations as input to the AO controller. These centroid locations were computed in Matlab for a user-defined aberration pattern, allowing us to test the ability of the DM to generate and compensate for various aberrations. We conclude that this device, in combination with another DM based on Micro-Electro Mechanical Systems (MEMS) technology, may provide better compensation of the higher-order ocular wavefront aberrations of the human eye

Shape memory polymers (SMPs) are attracting a great deal of interest in the scientific community for their use in applications ranging from light weight structures in space to micro-actuators in MEMS devices. These relatively new materials can be formed into a primary shape, reformed into a stable secondary shape, and then controllably actuated to recover their primary shape. The first part of this presentation will be a brief review of the types of polymeric structures which give rise to shape memory behavior in the context of new shape memory polymers with highly regular network structures recently developed at LLNL for biomedical devices. These new urethane SMPs have improved optical and physical properties relative to commercial SMPs, including improved clarity, high actuation force, and sharper actuation transition. In the second part of the presentation we discuss the development of SMP based devices for mechanically removing neurovascular occlusions which result in ischemic stroke. These devices are delivered to the site of the occlusion in compressed form, are pushed through the occlusion, actuated (usually optically) to take on an expanded conformation, and then used to dislodge and grip the thrombus while it is withdrawn through the catheter.

Rapid analysis of drugs in emergency room overdose patients is critical to selecting appropriate medical care. Saliva analysis has long been considered an attractive alternative to blood plasma analysis for this application. However, current clinical laboratory analysis methods involve extensive sample extraction followed by gas chromatography and mass spectrometry, and typically require as much as one hour to perform. In an effort to overcome this limitation we have been investigating metal-doped sol-gels to both separate drugs and their metabolites from saliva and generate surface-enhanced Raman spectra. We have incorporated the sol-gel in a disposable lab-on-a-chip format, and generally no more than a drop of sample is required. The detailed molecular vibrational information allows chemical identification, while the increase in Raman scattering by six orders of magnitude or more allows detection of microg/mL concentrations. Measurements of cocaine, its metabolite benzoylecgonine, and several barbiturates are presented.

In this work, the selective molecular recognition capability and high binding affinity of nucleic acid aptamers is integrated with the signal transduction methodology of molecular beacons for real-time monitoring of a protein target. An aptamer recognition element was modified to exist in a stem-loop configuration in the absence of a protein target, and in the presence of the protein target thrombin, the probe changes conformation. Upon binding to the target, this separation causes a physical separation of the attached fluorophore and quencher molecules, thereby allowing for an engineered, single- step recognition and signaling systems. The aptamer signaling probe was found to exhibit a maximum 12-fold change in signal when hybridized with a complement control, and a 3-fold change in signal with an excess of thrombin protein target. The fluorescence increased with increasing concentration of thrombin, until probe saturation where the fluorescence signal did not increase further, but leveled off in intensity. The signaling probe produced a rapid response, with 70% of the maximum signal achieved within a 15 second response time.

Optical properties of whole bovine blood are examined under conditions of different glucose loadings. Partial least-squares (PLS) is used to compute calibration models for glucose from spectra collected over the combination spectral region (5000 - 4000 cm^{- 1}) and first overtone - short wavelength spectral regions (9000 - 5400 cm^-1). These models achieve a prediction accuracy of approximately 1mM. Calibration models built for specific glucose absorption regions perform better than models generated strictly from the short wavelength region in which light scattering effects dominate. Net analyte signal (NAS) analysis is employed to investigate the spectral information that forms the basis for the models. The NAS reveals the portion of the glucose spectrum that is orthogonal to the spectral variance induced by the blood matrix. To investigate the selectivity of the spectral measurements, the glucose NAS is compared to residual absorbance spectra formed after subtraction of the non-glucose variance (estimated by application of principal component analysis to a set of blood samples with endogenous glucose concentrations). A match between the NAS and the residual spectra reveals that direct information associated with absorption of light by the glucose molecule is present in the measured data. A similar comparison is made with the regression vector associated with the PLS model. A match between the NAS and regression vector confirms that the correlations encoded in the calibration model do, in fact, arise from glucose absorption information. The results obtained through this work demonstrate that NAS analysis is a valuable tool for use in investigating the selectivity of multivariate calibration models.

Detection of multiple biologically relevant molecules was accomplished at sub-ng/mL levels in highly fouling media using fiber- optic based surface plasmon resonance sensors. Myocardial infarction markers, myoglobin and cTnI, were quantified in full serum with limits of detection below 1 ng/mL. Biologically relevant levels are between 15-30 ng/mL and 1-5 ng/mL for myoglobin and cTnI respectively. Cytokines involved in chronic wound healing, Interleukin 1, Interleukin 6, and tumor necrosis factor α, were detected at around 1 ng/mL in cell culture media. Preliminary results in monitoring these cytokines in cell cultures expressing the cytokines were obtained. The protein diagnostic of spinal muscular atrophy, survival motor neuron protein, was quantified from cell lysate. To obtain such results in complex media, the sensor's stability to non-specific protein adsorption had to be optimized. A layer of the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid is attached to the sensor. This layer optimizes the antibody attachment to the sensor while minimizing the non-specific signal from serum proteins.

In this paper, we describe a novel multiplexed surface plasmon resonance (SPR) sensor which is made of cyclic olefin copolymers (COCs, TOPAS^TM). This material has excellent chemical resistance, low water uptake (< 0.01%), and high refractive index (n_He-_Ne=1.53) suitable to use as an optical coupler (prism) as well as a sensor substrate. We fabricated a standard slide glass sized, prism integrated, and injection molded COC-SPR sensor which are being applied toward the multiplexed detection of DNA single nucleotide polymorphism (SNP). To evaluate the sensitivity of COC-SPR sensor, we first patterned MgF₂ on gold-coated COC-SPR sensor and observed the shift of minimum reflectivity (SPR dip) in pixel address. As incident light source we used an expanded, collimated, rectangular shaped He-Ne laser, with a diffuser for beam homogenization. With expanded laser beam we varied incident angle so that the angular shift is expressed as the darkest pixel shift on CCD. For optimized SPR characteristics and sensor configuration, analytical calculations (Fresnel equation) were performed, and the best SPR conditions were found to be d_Au~48 nm at wavelength λ=633 nm with respected resonance angle at θ_SPR =44.2° for COC-SPR sensor.

Saliva presents a minimally invasive alternative medium to blood for performing diagnostics¹. Microsphere sensors for ions, small organic molecules, and proteins are currently being developed and optical microarrays containing thousands of these sensors will be used for simultaneous multi-analyte analysis. The fiber bundle platform in use is 1mm in diameter and contains approximately 50,000 individually addressable 3.1μm fibers, each with an etched well capable of housing a single 3.1μm microsphere sensor. Micron-sized bead-based chemistries are produced in house, followed by deposition onto a fiber-optic bundle platform, allowing for multiplexed analysis. The ultimate goal is to develop a universal diagnostic system using saliva as the diagnostic medium. This platform will permit multiplexed analysis of a sample by integrating microfluidics with the optical arrays loaded with sensors capable of detecting relevant biomarkers associated with a wide range of disease states. Disease states that are currently under investigation include end stage renal disease (ESRD) and Sjoegrens Syndrome (SS).

The development of a compact structurally integrated platform for detection of multianalytes that consume oxygen in the presence of specific oxidase enzymes is described. The detection is based on monitoring the photoluminescence (PL) intensity or lifetime of a sensing element based on the oxygen sensitive dye Pt octaethyl porphyrin (PtOEP). The excitation source for the PL is an array of individually addressable green OLED pixels. The analytes are gas- phase and dissolved oxygen, glucose, lactate, and alcohol. The sensing element for each analyte includes a layer of PtOEP-doped polystyrene, whose PL lifetime decreases with increasing O₂ level, and a film or solution containing the oxidase enzyme specific to the analyte. Each sensing element is associated with two addressable ~2x2 mm² OLED pixels. The operation and performance metrics of the sensor under various conditions are described and discussed.

Immunoassays have been widely used in commercial, scientific and medical research for detection and quantification of analytes in complex mixtures. There is however a need for a point-of-care, multiplex diagnostic assays capable of providing rapid and quantitative measurements of analytes present in samples that are sufficiently simple to carry out without use of a laboratory or individuals trained in chemical analysis. We are developing a fluorescent lateral flow immunoassay platform to perform simultaneous, multiplexed detection of analytes in a complex fluid mixture along with instrumentation to optically quantitate the analytes in the sample. Our prototype imaging system is based on conventional 16-bit CCD optics, which enables the development of a rugged diagnostic instrument that can be further scaled down for point-of-care applications. We have compared protein microarrays with lateral flow assays (LFAs) to determine the sensitivity of each system for the measurement of distinct proteins in complex samples. We are pursuing the LFA platform such that it can easily be scaled to meet the requirements of any given screening application, and be implemented for use in a medical or surgical setting.

In this paper, the reflection resonance spectrum of a sub-wavelength diffraction grating-coupled waveguide is used to analyze biomolecular interactions in real time. When the diffraction grating waveguide structure is destroyed by external factors such as slight refractive index changes of the buffer or molecule adsorption on the grating surface, the optical path of the light coupled through the grating into the waveguide is changed and a resonance wavelength shift is induced as a result. By detecting this resonance wavelength shift, the optical waveguide biosensor provides the ability to identify the kinetics of the biomolecular interaction on an on-line basis without the need for the extrinsic labeling of the biomolecules. A theoretical analysis of the sub-wavelength optical waveguide biosensor is performed. A biosensor with a narrow reflection resonance spectrum, and hence an enhanced detection resolution, is then designed and fabricated. Currently, the detection limit of the optical waveguide sensor is found to be approximately 10^-5 refractive index units. The developed biosensor is successfully applied to study the kinetics of an antibody interaction with protein G adsorbed on the sensing surface.

The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.

Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

Single photon counting is the most sensitive method for detection of weak signals. However, it has rarely been used in DNA sequencing applications because of its complexity. We present a fiberized 16-channel single photon detection (SPD) module based on avalanche photo diodes (APD). The diodes are cooled at -20 °C and the average dark count is 700 c/s with APD operating at 10V over-voltage. The proposed system uses active quenching based on basic NAND gates and delay integrated circuits (ICs) and is compact in size, robust, easily portable and requires only 120Vac supply for its operation. The fiberization of the diodes using standard components allows the connection of this system to any other system using fibers with FC connectors with up to 400μm core diameters. Comparative performance with superior commercially available photon counting modules has been obtained. The SPD module has been successfully used for fluorescence detection of weak signals in a DNA sequencing instrument and hence the suitability of large active area APD model for DNA sequencing has been verified experimentally.