This paper describes the fabrication and characterization of the analytical properties of fluorescence-based zinc ion sensing glass slides and antibody based zinc sensors and their application in monitoring zinc release from beta pancreatic cells. The zinc ion indicator ZnAF-2 {6-[N- [N', N'-bis (2-pyridinylmethyl)-2-aminoethyl] amino-3',6'-dihydroxy-spiro[isobenzofuran-1(3H),9'-[9H] xanthene]-3-one} was modified to include a sufficiently long linking aliphatic chain, with a terminal carboxyl functional group. The activated carboxyl-modified ZnAF-2 was conjugated to the amino silanized surface of glass slides and to free amino groups of the A2B5 antibody molecules. The sensors were used to monitor zinc ion release events from glucose-stimulated pancreatic cells.

Surface-Enhanced-Raman-Spectroscopy (SERS) is potentially a very sensitive technique for the detection of biological agents (i.e., proteins, viruses or whole cell bacteria). However, since initial reports, its utility has not been realized. Its limited acceptance as a routine analysis technique for both chemical and biological agents is largely due to the lack of reproducible SERS-active substrates. Most established SERS substrate fabrication schemes are based on selfassembly of the metallic (typically, Au, Ag, Pt, Pd or Cu) surfaces responsible for enhancement. Further, these protocols do not lend themselves to the stringent control over the enhancing feature shape, size, and placement on a nanometer scale. SERS can be made a more robust and attractive spectroscopic technique for biological agents by developing quantifiable, highly sensitive, and highly selective SERS-active substrates. Recently, novel SERS-active substrates, fabricated from nano-patterned Si and Au have been commercialized and are easily obtained in the marketplace. Commercialized Au SERS-active substrates fabricated using semiconductor manipulation and routine metal vapor deposition techniques used for the spectral analysis of intact bacterial cells. This talk will focus on the substrate characterization (microscopic and spectral) and application towards whole cells.

We have developed novel surface enhanced Raman scattering (SERS)-based nanoimaging probes for dynamic chemical imaging in a non-scanning format. These probes have been developed for obtaining sub-diffraction limited chemical measurements of various biochemical species (e.g., lipids, proteins, etc.) as well as biological organisms (e.g., bacteria, etc.). They combine qualitative and quantitative information obtained from SERS with the imaging capabilities of coherent fiber optic bundles. Nanoimaging probes are fabricated from coherent fiber optic bundles composed of 30,000 individual 4 μm diameter fiber elements. Using a CO₂ laser based micropipette puller, bundles are tapered on one end resulting in the formation of equi-diameter individual elements tens of nanometers to hundreds of nanometers in diameter. Employing these probes, inherent image magnification and submicron spatial resolution is possible. Across these tapered probe tips, uniformly roughened surface features are creating by HF acid etching. These surface features consist of six cladding peaks that surround each individual fiber elements' core, which are uniform in size, shape, structure, and spacing. SERS active surfaces are created on the tapered tips of the probes by selectively depositing silver onto these cladding peaks, creating an array of highly ordered uniform silver islands across these nanoimaging probes' surfaces. This fabrication process results in a high degree of uniformity in SERS enhancement across the image surface of the probes (< 3.0% RSD), which is essential for reproducible quantitative imaging applications. Further SERS enhancement and specific excitation wavelength tuning can be achieved by controlling the spacing between silver islands and the overall size of the silver islands uniformly arrayed across these SERS probe tips. Characterization and imaging of biochemical species and model compounds using these SERS nanoimaging probes is presented in this manuscript.

This paper presents a novel intrinsic fiber optic Fabry-Perot (FP) structure with a micrometric diameter tip. With the FP cavity inside the fiber, the change in optical path length difference (OPD) caused by the environment can be demodulated. With such a tiny protrusion, the sensor can be inserted into micron size cells for intracellular measurements. This label-free detection method is very useful in biological areas such as DNA hybridization detection. It provides a valuable tool for intracellular studies that have applications ranging from medicine to national security. In addition, the fabrication is simple including only cleaving, splicing, and etching. The signal is stable with high visibility. Last but not the least, the structure shows great promise for reduction to nanometric size. Once this goal is achieved, the sensor can be inserted into most cells with minimal invasiveness.

Based on high performance fast tunable phase retarder and novel algorithm, an innovative polarization imaging solution is proposed. It allows very fast recording the polarization images at the speed limit of a CCD. It contains no moving parts and can accommodate to most of the existing CCD cameras. The unique measurement procedure allows efficient, accurate sensing of the polarization imaging. A computer-aided diagnosis software has been developed for the proposed polarization imaging system.

Trans-cellular calcium currents play a central role in the establishment of polarity in differentiating cells. Typically these currents are measured and studied experimentally using ion selective glass microelectrodes. We have recently developed an in silico cell electrophysiology lab-on-a-chip device with the specific science objectives of measuring these transcellular calcium currents in an advanced throughput format. The device consists of 16 pyramidal pores on a silicon substrate with four Ag/AgCl electrodes leading into each pore on the four poles. An SU-8 layer is used as the structural and insulating layer and a calcium ion selective membrane is used to impart ion selectivity to the Ag/AgCl electrodes. In this paper we demonstrate the utility of the cell electrophysiology biochip in measuring these transcellular calcium currents from single cells using the model biological system Ceratopteris richardii. We monitored these fern spores during germination and pharmacologically inhibited biophysical calcium transport. These results demonstrate the utility and versatility of the in silico cell electrophysiology biochip. While this version of the biochip was engineered to fulfill the specific science objectives of measuring trans-cellular calcium currents from Ceratopteris fern spores, the chip can easily be modified for a variety of biomedical and pharmacological applications. Future

Surface Plasmon Resonance (SPR) spectroscopy is a potentially valuable tool for measuring protein-protein interactions and protein levels in vitro and in vivo. Fiber-optic based SPR allows for rapid quantitative measurement of disease markers such as the truncated (exons 1-7) survival of motor neuron (SMN) protein. Unlike micro fluidics-based SPR systems, sample loss is eliminated in fiber-optic SPR and the small size of the fiber optic probes (400μm or smaller) facilitates the potential for use in vivo. Recombinant SMN protein overexpressed in E. Coli as well as native SMN from cultured HeLa cells has been successfully measured using fiber-optic SPR.

Raman spectroscopy is a powerful technique for rapid, non-invasive and reagentless analysis of materials, including biological cells and tissues. Raman Molecular Imaging combines high molecular information content Raman spectroscopy and digital full field imaging to enable the investigation of cells and tissues. We have conducted widefield imaging using a new class of birefringent liquid crystal tunable filter that provides high throughput over an extended wavelength range. This tool has been applied to investigate the linkage between reagentless spectral imaging in tissue and cells and standard reagent based approaches. In this report, we describe Raman imaging data on a clinical tissue sample and cultured cells. The results demonstrate the sensitivity of Raman Molecular Imaging and fluorescence spectral imaging to molecular differences in biological systems laying the foundation for the eventual use of this approach as a biological research and clinical diagnostic tool.

The biomolecular interactions between a fluorescently labeled aptamer and whole cell Campylobacter jejuni(C. jejuni) have been characterized using capillary electrophoresis with laser-induced fluorescence detection. From electrophoretic analysis, the bound complex forms, unbound aptamer, and cells were visualized. The relative binding affinity of the DNA aptamer with C. jejuni was compared with other food-borne pathogens including Escherichia coli O157:H7 and Salmonella typhimurium. Preliminary data suggests that this aptamer exhibits strong binding affinity towards C. jejuni with minimal cross reactivity over other food pathogens when equivalent cell concentrations were used.

A detailed study of SERS enhancements from novel multilayer gold film over nanoparticle (GFON) substrates is presented. These multilayer GFON substrates were optimized in terms of the number of metal layers, and the amounts of gold and silver oxide deposited. These multilayer GFON substrates were also structurally characterized in terms of surface roughness. No significant changes in the surface roughness of these multilayer GFON substrates, even with different layers of gold, have been observed, suggesting there is no direct correlation between the multilayer SERS enhancements and the surface roughness. UV-Vis reflectance spectra of these substrates were also characterized, indicating that the significant multilayer enhancements require the presence of silver oxide layers separating the continuous gold film layers.

A real-time practical fiber micro-drop sensor, which can determine the physical and chemical parameters of the liquid, has been designed and realized. Liquid drops are formed at the tip of a liquid sensing head passing thin tubing. The infrared light is injected into the drop through an optical fiber and is received by another fiber after reflection, scattering and absorption with the liquid drop. The curve of the light intensity vs. time is called as Fiber Fingerprint Drop Trace (FFDT), which depends on the characteristics of the liquid to be tested. The trend and distinguishing feature of the FFDT is strongly relative to the shape-variation of the liquid drop. The correlation methods were induced to the signal analysis process.

Label-free optical biosensors offer advantages for many applications due to their simplicity and low cost compared to fluorescence detection. Thus, it is desirable to develop label-free sensors that can be integrated with advanced microfluidic systems into dense, multi-purpose biosensor arrays. One candidate technology is ring resonators, which utilize the resonating whispering gallery modes to create a strongly enhanced optical field in the sensing volume. Because of the high Q-factor of ring resonators, the optical field can be enhanced by 2-3 orders of magnitude, which leads to much smaller required light-matter interaction length and sensing volume. These are critical characteristics for dense integration into lab-on-a-chip systems. We have developed a novel label-free ring resonator sensor based on a liquid core optical ring resonator (LCORR). This system uses a glass capillary as both the fluidics and the ring resonator. With the LCORR, we have demonstrated a measurable whispering gallery mode spectral shift of 30 pm/refractive-index-unit (RIU), which leads to a detection limit of approximately 10^-6 RIU. Additionally, we have achieved an estimated detection limit for protein molecules of 10 pg/mm². These experimental demonstrations of this novel sensing system will lead to the development of highly sensitive label-free sensors that are well-suited for dense integration with advanced microfluidics for lab-on-a-chip systems.

A polymer based biochip for rapid 2-D separations of peptides, proteins, and other biomedically relevant molecules was designed and fabricated. Like traditional 2D polyacrylamide gel electrophoresis (2D-PAGE) methods, the device will allow molecules to separate based on isoelectric point (pI) and molecular weight (MW). The design, however, integrates both an initial capillary isoelectric focusing (cIEF) step followed by capillary electrophoresis (CE) in multiple parallel channels, all on a single microfluidic chip. Not only is the "lab-on-a-chip" design easier to use and less expensive, but the miniaturization of the device produces very rapid separations, on the order of seconds. Fluorescence detection will be used in the preliminary stages of testing, but the device is also equipped with integrated electrodes in the electrophoresis channels to perform multiplexed electrochemical detection for quantitative analysis. We present the chip design and fabrication, as well as the initial test results demonstrating cIEF and CE with one analyte. Furthermore, we introduce preliminary work on the use of a polyacrylamide gel in the electrophoresis channels.

The BioCD is a class of self-referencing interferometric optical biosensor that measures phase modulation from proteins on a spinning disk. The optical detection of the patterns at high speed yields low noise floors far from 1/f noise. Two scans of a disk before and after a 20 hour buffer wash are differenced yielding an rms surface height measurement error of 45 pm corresponding to 5 femtograms of protein within a focal spot diameter. Simple area scaling relations are derived that predict the performance of immunoassays as a function of well area. The scaling mass sensitivity of the BioCD is determined to be 0.25 pg/mm under the conditions of an assay, with a metrology limit of the technique between 0.05 to 0.1 pg/mm. The BioCD sensitivity is equivalent to the best reported surface mass sensitivity of surface plasmon resonance sensors, and is achieved without resonant structures and hence is easy to fabricate and operate.

Injection molding technology offers the most competitive potential to meet the growing demand for cost-effective manufacturing of components with micro and nanoscale features due to its far greater production rates than the other techniques. Since conventional mold tooling materials and techniques are not suitable for sub-micron scale molding, mature silicon processing technology were evaluated as tooling for these features. Simple pattern geometries of trench lines were employed to simplify the analysis and all parts were molded using optical grade high-flow polycarbonate. Replication quality was evaluated in terms of depth ratio (height of molded feature/depth of corresponding tooling feature) and root-mean-square roughness. Although perfect replication has not been achieved with the given system, several factors including surface adhesion and feature aspect ratio were found to be critical for replication of nanoscalefeatures. Of four factors possibly affecting replication, adhesion of the polymer to silicon surface during ejection was found to be critical and is influenced by processing temperatures, cooling times, tooling mounting systems, and tooling surface roughness. Trapped of air in tooling trenches, damage to the silicon tooling during molding, and shrinkage of polymer during cooling may also have contributed to less-than-perfect replication. All factors seem synergistic and the effects are greater for small feature geometries.

This paper presents experimental and theoretical studies of time-gated discrimination of long-lived luminescence (lifetime: 1~2000μs) labelled target-organisms against non-target autofluorescence background (lifetime: <100 ns) in flow cytometry. A theoretical model of such a TGL flow cytometer is developed which takes account of flow speed, illumination and detection apertures, fluorescence label lifetime, and pulsed illumination and gated detection timing sequences. Ultraviolet LED and channel photomultiplier were found to be practical as pulsed excitation sources and gated detector for TGL flow cytometry. The prototype cytometer was constructed and optimized to operate at 6 k Hz repetition rate of TGL cycles consisting of 100 μs LED excitation and ~60 μs gated detection. The spatial counting efficiency was evaluated by enumerating 5.5 μm diameter europium microspheres resulting in a counting accuracy approaching 100%.

We demonstrate the improvement of fluorescence immunoassay (FIA) diagnostics in deploying a newly developed compact diode-pumped solid state (DPSS) laser with emission at 315 nm. The laser is based on the quasi-three-level transition in Nd:YAG at 946 nm. The pulsed operation is either realized by an active Q-switch using an electro-optical device or by introduction of a Cr⁴⁺:YAG saturable absorber as passive Q-switch element. By extra-cavity second harmonic generation in different nonlinear crystal media we obtained blue light at 473 nm. Subsequent mixing of the fundamental and the second harmonic in a β-barium-borate crystal provided pulsed emission at 315 nm with up to 20 μJ maximum pulse energy and 17 ns pulse duration. Substitution of a nitrogen laser in a FIA diagnostics system by the DPSS laser succeeded in considerable improvement of the detection limit. Despite significantly lower pulse energies (7 μJ DPSS laser versus 150 μJ nitrogen laser), in preliminary investigations the limit of detection was reduced by a factor of three for a typical FIA.

Fourier domain optical coherence tomography (FD-OCT) is an interferometric imaging technique that allows imaging to depths of a few mm in scattering biological tissues with high resolution of the order of 1-10 μm. However, the usefulness of FD-OCT is limited by background and autocorrelation interference terms that reduce the sensitivity and by phase ambiguity that halves the useful imaging depth range. These limitations can be overcome by obtaining the full, complex spectral interferogram. Simultaneous detection of the imaginary and real terms is obtained by phase modulating the reference arm of the interferometer and detecting at the first and second harmonics. A mathematical derivation of harmonically detected FD-OCT and experimental measurements showing that phase ambiguity artifacts can be suppressed by up to 70 dB are presented. The method provides efficient suppression of the complex conjugate, dc, and autocorrelation artifacts and has low sensitivity to phase noise. Beyond the removal of artifacts, the ability to obtain the full, complex interferogram is key to the development of spectrally resolved FD-OCT which would add depth-resolved spectroscopic detail to the structural information.

Identifying injuries, deformities, and diseases by non-invasive instrumental means has been a major innovation in medicine. Diagnostic and imaging medical devices have revolutionized diagnosis and surgery, providing more efficient way to identify injuries and diseased or damaged tissues. In this paper, identification of different animal tissues using a miniature near-infrared (NIR) spectrometer will be demonstrated. Each tissue type contains different amounts of moisture and proteins, and by using this miniature spectrometer, a miniature fiber-optic probe and chemometrics; the ability to recognize tissues spectral differences is established.

Deep Illumination Angular Domain Imaging employs a micromachined angular filter array to detect photons emitted from the scattered light created by a laser source aimed deep beneath the turbid medium surface. As this source light is scattered, a ball of illumination is formed within the medium. This deep illumination source emits scattered light in all directions and illuminates objects near the surface from behind. When photons from this illumination ball pass an object and reach the angular filter, light that was not subsequently scattered, passes through to a camera detector whereas scattered photons are rejected by the filter. The angular filter consists of an array of high-aspect ratio channels fabricated via silicon bulk micromachining. Under illumination by an argon ion (488-514 nm) laser, two-dimensional phantom test objects were observed in high scattering media up to 3 mm deep in the medium at effective scattering coefficients, μ_seff up to 5.8 cm-1. Scan results are reconstructed and enhanced using various image processing techniques to enhance the spatial image resolution and image contrast and to reduce noise.

Optically transduced sensors (optrodes, or optodes) offer significant advantages over polarographic techniques for measuring oxygen. In biology and medicine, how we make measurements is very important, and this is especially true in terms of physiological exchange. Cellular and tissue oxygenation is a function of background concentration and respiratory demand, and in pure physical terms this is best expressed in terms of molecular flux based on Fick's law. Measuring dynamic flux from biological systems requires sensing technology that can measure activity in multiple dimensions. Here we report the development of a self-referencing oxygen optrode (SRO) for reliably making noninvasive measurements of oxygen flux from a variety of biological systems. The self-referencing microsensor technique was adapted to operate optrodic oxygen sensors through the integration of optical sensing instrumentation with software-controlled data acquisition and micro-stepping motion control. This allows the sensor to scan biologically active gradients of oxygen flux directly, as it relates to cellular and tissue respiratory activity. The technique was validated first using artificially generated oxygen gradients, which are theoretically modelled and compare with measured signals. Subsequently, the SRO was applied in basic research applications to non-invasively measure molecular oxygen flux from a variety of animal and plant systems.

Patient motion during single photon emission computed tomographic (SPECT) acquisition causes inconsistent projection data and reconstruction artifacts which can significantly affect diagnostic accuracy. We have investigated use of the Polaris stereo infrared motion-tracking system to track 6-Degrees-of-Freedom (6-DOF) motion of spherical reflectors (markers) on stretchy bands about the patient's chest and abdomen during cardiac SPECT imaging. The marker position information, obtained by opposed stereo infrared-camera systems, requires processing to correctly record tracked markers, and map Polaris co-ordinate data into the SPECT co-ordinate system. One stereo camera views the markers from the patient's head direction, and the other from the patient's foot direction. The need for opposed cameras is to overcome anatomical and geometrical limitations which sometimes prevent all markers from being seen by a single stereo camera. Both sets of marker data are required to compute rotational and translational 6-DOF motion of the patient which ultimately will be used for SPECT patient-motion corrections. The processing utilizes an algorithm involving least-squares fitting, to each other, of two 3-D point sets using singular value decomposition (SVD) resulting in the rotation matrix and translation of the rigid body centroid. We have previously demonstrated the ability to monitor multiple markers for twelve patients viewing from the foot end, and employed a neural network to separate the periodic respiratory motion component of marker motion from aperiodic body motion. We plan to initiate routine 6-DOF tracking of patient motion during SPECT imaging in the future, and are herein evaluating the feasibility of employing opposed stereo cameras.

It is estimated that 750,000 cases of severe sepsis occur in the United States annually, at least 225,000 of which are fatal, resulting in significant utilization of healthcare resources and expenses. Significant progress in the understanding of pathophysiology and treatment of this condition has been made lately. Among the newer treatment strategies for critically ill patients are the administration of early goal directed therapy, and Recombinant Human Activated Protein C (Drotrecogrin alfa (activated) [DTAA]) for severe sepsis. However, mortality remains unacceptably high.

Extended weightlessness causes numerous deleterious changes in human physiology, including space motion sickness, cephalad fluid shifts, reduced immune response, and breakdown of muscle tissue with subsequent loss of bone mass and formation of renal stones. Furthermore, these physiological changes also influence the metabolism of drugs used by astronauts to minimize these deleterious effects. Unfortunately, the changes in human physiology in space are also reflected in drug metabolism, and current pre-flight analyses designed to set dosage are inadequate. Furthermore, current earth-based analytical laboratory methods that employ liquid or gas chromatography for separation and fluorescence or mass spectrometry for trace detection are labor intensive, slow, massive, and not cost-effective for operation in space. In an effort to overcome these instrument limitations we have been developing a sampling device to both separate these drugs and metabolites from urine, and generate surface-enhanced Raman (SER) spectra. The detailed molecular vibrational information afforded by Raman scattering allows chemical identification, while the surface-enhancement increases sensitivity by six or more orders of magnitude and allows detection of nanogram per milliliter concentrations. Generally no more than 1 milliliter of sample is required and complete analysis can be performed in 5 minutes using a portable, light-weight Raman spectrometer. Here we present the SER analysis of several drugs used by astronauts measured in synthetic urine and reconstituted urine.

A hollow optical fiber Raman probe equipped with a ball lens is developed to study the stomach cancer. The probe generates little Raman scattering or fluorescence noise because the HOF confine light into the air core. In order to maximize the collection efficiency and the spatial resolution, material and size of the ball lens is optimized. The total diameter of the probe head is 0.64 mm. The probe is useful for measurement of layered sample by the contact mode because it has very short focal length and high depth resolution. In vivo experiments in a stomach of a living rat demonstrate the high viability of the probe for noninvasive diagnosis of the stomach cancer.

A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

A novel Surface Plasmon Resonance (SPR) sensor to detect glucose using molecularly imprinted polymer (MIP) will be presented in this paper. SPR has been traditionally used as a probe for surface interaction of large molecules but harder to measure small molecules since the effective change in the SPR condition becomes smaller. The accurate measurement of glucose in complex physiological fluids like urine is particularly challenging since the constituents of these fluids vary significantly from person to person and even throughout the day for a particular individual. The polymer was prepared by crosslinking polyallyamine in the presence of Glucose Phosphate, monobarium salt (GPS-Ba) and attached to a 50 nm thin film of gold which had been sputtered on top of a glass slide, via amide coupling. Upon removal of the template, this sensor was used to detect glucose in human urine in physiologically significant levels (1-20 mg/ml). Enhancement of the glucose sensor was made possible by incorporating gold nanoparticles which improved the signal. This study has demonstrated the specific detection of glucose in a complex physiological fluid using SPR spectroscopy. The association of glucose to the imprinted polymer results in the swelling of the polymer that can be tracked by the minima in SPR spectra. The sensitivity of this method, while lower than protein based detection schemes, is sufficient for quantitative measurement of glucose in urine at physiologically significant levels without extensive pre-treatment of the sample. Given the nature of the weak non-covalent binding of glucose to the amine functional groups, the scheme used here can be adapted to detect a number of different molecular species of sizes comparable to that of glucose without the need for extensive sample preparation or use of chemicals with limited shelf life.