This paper describes the development of novel particle-based fluorescence resonance energy transfer (FRET) biosensors. It describes the fundamentals of FRET in heterogeneous systems and the application of the new sensors in monitoring the binding affinity of carbohydrates and glycoproteins to lectins, which are carbohydrate binding proteins. The sensing approach is based on FRET between fluorescein (donor) labeled lectin molecules, adsorbed on the surface of micrometric polymeric beads, and polymeric dextran molecules labeled with Texas Red (acceptor). The FRET signal of the sensor decreases in the presence of carbohydrates or glycoproteins that inhibit the binding of Texas Red-labeled dextran molecules to the lectinic binding sites. The new FRET sensors could discriminate between carbohydrates and glycoproteins based on their binding affinity to the FRET sensing particles. Thery were also used for quantitative analysis of carbohydrates and glycoproteins in aqueous samples.

The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

We have developed and optimized novel nanosphere-based silver coated SERS substrates for the detection of proteins. These SERS substrates were optimized for silver thickness, number of silver layers, and extent of silver oxidation between layers. Immuno-nanosensors capable of being inserted into individual cells and non-invasively positioned to the sub-cellular location of interest using optical tweezers were constructed from monodisperse silica nanospheres. Silica nanospheres ranging in diameter from 100 to 4500 nm were condensed from tetraalkoxysilanes in an alcoholic solution of water and ammonia. By varying the reaction conditions, accurate control of the silica nanospheres’ diameter was achieved. Silica sphere sizes were optimized for SERS signal response. Nanosphere-based SERS substrates were made by depositing multiple layers of silver on the nanospheres, followed by binding of the antibody of interest to the silver. In binding the antibodies, different crosslinkers were characterized and compared. On one end, each of these crosslinkers contained sulfur or isothiocyanate groups which bound to the silver surface, while the other end contained a carboxylic or primary amine group which reacted readily with the antibodies. In order to evaluate these substrates, SERS spectra of different proteins, such as insulin and interleukin-2 (IL-2), were obtained. By using silver, as the metal surface for SERS, red and near-infrared excitation wavelengths (i.e., 600-700 nm) can be used. Excitation in this range helps to avoid photodamage to cells and reduces any autofluorescence background. Evaluation of these SERS substrates was performed using a 10 mW HeNe laser, operating at 632.8 nm, in a collinear excitation/detection geometry. The SERS signals were filtered with a holographic notch filter, dispersed by 1/3 meter spectrometer and detected using an intensified charge coupled device (ICCD). This paper discusses the fabrication and optimization of these nanosensors, as well as their potential applications.

We report results of our recent efforts to develop nano-tools to study proteins and their interactions in complex environments that exist on the cell membrane and inside the cells. Due to the spatial constraints imposed on the mobility of cell constituents, it is reasonable to expect that the nature and dynamics of the biomolecular interactions in a living cell would be substantially different from those routinely observed in dilute solutions. Nanotechnology has begun to provide tools with which to monitor processes that occur in membranes and intracellular regions. Nano-optics is a rich source of such emerging tools. Tapered optical fibers coated with metallic films can effectively confine excitation light to sub-wavelength linear dimensions and cubic nanometer excitation volumes. This leads not only to a resolution that exceeds the diffraction-limited values, but also to the elimination of the background signal. Thus, highly localized and specific regions of cellular function can be investigated. By immobilizing silver colloidal nanoparticles on such tapered fibers we have also fabricated surface enhanced Raman scattering (SERS) probes. Nanoprobes have been found to enable detection of fluorescent antibody molecules immobilized on a functionalized glass surface and polychromic quantum dots in picomolar solutions. In addition, we have successfully inserted nanoprobes with dimensions of 30-80 nm into both adherent insect and mammalian cells with maintenance of their viability. We summarize our development of optical nanoprobes with the motivation to detect cell-surface and intracellular proteins of the interleukin-5 system in native cellular environments, through quantum dot fluorescence and SERS.

DNA damage is caused by a variety of foreign and endogenous compounds. There are endogenous photosensitizers in cells, such as porphyrins and flavins, which may create damage in the presence of UV-A light. Typically, samples are analyzed by ³²P-postlabelling and electrophoretic separation or by LC-MS separation and detection. Separation by HPLC is common; however, in all instances, the DNA sample is hydrolyzed down to nucleosides prior to analysis. It will be shown here that ion-pairing reversed phase high performance liquid chromatography (IP-RPLC) has the ability to provide biophysical information concerning the sites of UV-A induced photosensitizer damage on an intact oligonucleotide concurrent with the separation. IP-RPLC is less labor intensive and faster than electrophoretic methods and it is less costly than LC-MS. IP-RPLC can also be used to purify modified oligonucleotides for further use and analysis. This technique is sensitive to the charge, conformation, and sequence characteristics of the nucleic acid sample and may be used to determine the damage or modifications made to DNA by a variety of compounds.

With the increasing interest in simultaneous detection of specific DNA hybridization events, the development of methods to measure multiple DNA interactions at one time is of great importance. Conventional microarrays allow thousands of DNA hybridization interactions to be measured at once, however, this method of detection is limited by high cost as well as the stability and characteristic properties of fluorescent dyes. Here, barcoded nanowires are investigated as replacements for fluorophores on glass surfaces such as those used in microarrays. Potential advantages of nanowires include ease of reflectance-based optical read-out, the large number of tags available, and ability to distinguish multiple hybridizations occurring in a single DNA spot. A method of attaching DNA to glass microscope slides was developed which includes the use of a carboxy terminated silane to derivatize glass slides for DNA attachment. Also determined here is the efficiency of using nanowires as tags in complementary DNA hybridization events. An average of ~5% nonspecific binding was reported for nanowire attachment for all samples.

Application of the new platform of structurally integrated luminescent chemical and biological sensors, in which the photoluminescence (PL) excitation source is an organic light-emitting device (OLED), is demonstrated for the detection of oxygen, glucose, hydrazine, and anthrax lethal factor (LF). The oxygen sensors are based on the collisional quenching of the PL of tris(4,7-diphenyl-1,10-phenanthroline) Ru (II) (Ru(dpp)) and Pt octaethyl porphyrin (PtOEP) by O₂. The glucose sensors are based on the O₂ sensors, to which glucose oxidase, which catalyzes the reaction between glucose and O₂, is added. The oxygen and glucose sensors are operable in either the PL intensity I mode or the PL lifetime t mode, where the value of I or t yields the oxygen level. In the t mode, the need for sensor calibration, which remains a challenge in real-world sensing applications, is eliminated. The performance of sensors based on [blue 4,4'-bis(2,2'-diphenylvinyl)-1,1'-biphenyl (DPVBi) OLEDs]/[Ru(dpp)] are compared to those of [green tris(8-hydroxy quinoline) Al (Alq₃)]/[PtOEP]. The latter are strongly preferred over the former, due to the relatively long t of PtOEP (~130 ms in the absence of O₂), and the higher efficiency and brightness of the green Alq₃ OLEDs. Demonstration of the hydrazine sensor is based on the reaction between nonluminescent anthracene-2,3-dicarboxaldehyde and hydrazine or hydrazine sulfate, which generates a luminescent product. The anthrax LF sensor is based on the cleavage of certain peptides by the anthrax-secreted LF enzyme. As the LF cleaves a fluorescence resonance energy transfer (FRET) donor-acceptor pair-labeled peptide, and the two cleaved segments are separated, the PL of the donor, previously absorbed by the acceptor, becomes detectable by the photodetector.

Chemotherapy drug dosage is based on the limited statistics of the response of previously treated patients and administered according to body surface area. Considerably better dose regulation could be performed if the drug metabolism of each patient could be monitored. Unfortunately, current technologies require multiple withdrawals of blood to determine metabolism, a precious fluid in limited supply. Saliva analysis has long been considered an attractive alternative, but unfortunately standard techniques require large quantities that are difficult to obtain. In an effort to overcome this limitation we have been investigating the ability of metal-doped sol-gels to both separate drugs and their metabolites from saliva and generate surface-enhanced Raman spectra. Surface-enhanced Raman spectroscopy has the potential to perform this analysis with just a few drops of sample due to its extreme sensitivity. Preliminary measurements are presented for the chemotherapy drug, 5-fluorouracil, and its two metabolites 5-fluorouridine and 5-fluoro-2'-deoxyuridine, and the potential of determining metabolism on a patient-by-patient basis.

Surface-Enhanced-Raman-Scattering (SERS) is potentially a very sensitive spectroscopic technique for the detection of biological agents (i.e., proteins, viruses or bacteria). However, since initial reports, its utility has not been realized. Its limited acceptance as a routine analysis technique for biological agents is largely due to the lack of reproducible SERS-active substrates. Most established SERS substrate fabrication schemes are based on self-assembly of the metallic (typically, Au, Ag, Pt, Pd or Cu) particles 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. Electron Beam Lithography (EBL), a semiconductor fabrication technique, can be utilized to address many of the obstacles which have limited the broad acceptance of SERS. Specifically, EBL can be employed to precisely control the shape, size and position (on a nanometer scale) of the SERS substrate enhancing features. Since Ashkin's seminal work in the early 1970s, the optical trapping phenomenon has been broadly accepted as a powerful tool to study micrometer-scale biological particles. Recently, research in our laboratory has demonstrated that it is possible to combine the Optical Trapping phenomenon and SERS to develop a high sensitivity spectroscopic technique for the detection of individual bacterial spores. Highly reproducible SERS-active substrates fabricated using EBL have been utilized with this novel spectroscopic technique to investigate the utility of SERS technique for the spectral discrimination of bacterial spores. The SERS substrate fabrication methodology, substrate reproducibility and SERS spectral reproducibility will be discussed.

We have developed and characterized novel multilayered metal film-based surface-enhanced Raman scattering (SERS) substrates capable of enhancing SERS signals over an order of magnitude compared to conventional single layer substrates. In addition to enhanced signal intensity, these multilayered metal film substrates also exhibit longer SERS active lifetimes, higher reproducibility and lower detection limits than single layer silver substrates. Multilayered metal film substrates were fabricated by repeated vapor deposition of metal films over nanometer sized silica spheres. Different sizes of silica spheres were evaluated in order to obtain the optimal SERS enhancements. Meanwhile, different coating methods, drop coating and spin coating, were applied to form silica sphere layers that provided the roughness for SERS enhancements. These two coating methods were also compared for various silica sphere sizes by investigating their effects on the SERS enhancements. By applying additional silver layers on top of silver film over silica sphere SERS substrates, multi-layer enhancements can be observed. Additionally, different metals, such as gold, were used to further optimize the stability and reproducibility of these novel substrates. In order to speed up the fabrication of these multiple metal layer SERS substrates, silver oxide layers produced in an oven were investigated, reducing fabrication time by a factor of 50.

Chemical imaging is a powerful technique combining molecular spectroscopy and digital imaging for rapid, non-invasive and reagentless analysis of materials, including biological cells and tissues. Raman chemical imaging is suited to the characterization of molecular composition and structure of biomateials at submicron spatial resolution (< 250 nm). As a result, Raman imaging has potential as a routine tool for the assessment of cells and subcellular components. In this presentation, we discuss Raman chemical imaging and spectroscopy of single human cells obtained from a culture line. Rapid three dimensional Raman imaging is shown using deconvolution to improve image quality.

We have developed a surface enhanced Raman scattering (SERS) based nanoimaging probe capable of chemical imaging with nanometer scale spatial resolution. Using this SERS-nanoimaging probe it is possible to image individual chemical components within sub-cellular environments. The probe consists of a tapered coherent fiber optic imaging bundle that has been coated with a roughened layer of metal, providing a SERS active substrate. The fiber optic bundle is tapered using a specially programmed micropipette puller, allowing precise control over the probe tip's diameter, and thus the resolution of images. Tapered bundles having individual fiber elements ranging from 100-800 nanometers on the tapered end and 4 micrometers in diameter on the proximal end have been investigated. Through modification of the fibers' tapered tips, generation of nanoscale imaging with inherent image magnification and short pass filtering effects is possible. Following tapering of the fiber optic bundles, the fiber probes are spin-coated with alumina particles and coated with silver to provide a reproducible SERS active surface. Characterization of the response of these SERS nanoimaging probes has been evaluated using common SERS active chemical species (e.g., benzoic acid, brilliant cresyl blue, etc.) and application of these nanoimaging sensors to biological systems is discussed.

Interfacing organisms and hardware is a promising and challenging research frontier. The threat of biological weapons and the rising cost of health care have pushed detection of pathogens and their toxins to the forefront of that frontier. Both the military and the civilian sectors require that this detection be fast, accurate, sensitive, and inexpensive. We describe an electrochemical detection method that relies upon "molecular-scale gates" capable of being activated by a biological agent. We discuss our most recent experimental and modeling results, which take into account DNA folding and introduce the concept of tethering to boost the detection signal. Preliminary results show dramatic and specific recognition of target molecules.

Progress in the application of vertically-aligned carbon nanofibers (VACNF) as parallel subcellular and molecular-scale probes for biological manipulation and monitoring is reported. VACNFs possess many attributes that make them very attractive for implementation as functional, nanoscale features of microfabricated devices. For example, they can be synthesized at precise locations upon a substrate, can be grown many microns long, and feature sharp, nano-dimensioned tips. This, and their needlelike, vertical orientation upon a substrate, makes them particularly attractive as multielement cellular scale probes or as a parallel embodiment of traditional single-point microinjection or microelectrophysiological systems. We will overview our progress with fabricating and characterizing several embodiments of VACNF cell probing systems, which all feature arrays of nanoscale electrochemically-active probing regions at the tips of individually electrically-addressed nanofiber elements. We also overview our techniques of integrating these probing structures with intact cells and how these structures may be used on a massively parallel basis for measurement and control around and within viable cells.

High-performance liquid chromatography with ultra violet and photo-assisted electrochemical detection (HPLC-UV-PAED) has been applied to the sensitive and selective determination of organic nitro compounds. The system was first developed for the determination of nitro explosives, and PAED has shown superior sensitivity over UV detection for these compounds (i.e., <1 part-per-trillion for HMX). The system also shows enhanced selectivity over the traditional UV method in that two detectors can be used for improved analyte identification. Also, having two detectors permits chemometric resolution of overlapping peaks, and this is not addressed in the UV method. Because this method is applicable to a wide range of nitro explosives, it was predicted that PAED would show the same sensitivity and selectivity toward other types of nitro compounds. Since its development, the system's use has been expanded to include the determination of nitro-containing pharmaceuticals and glycosylated nitro compounds in biological matrices. Model compounds were chosen, specifically nitroglycerin and related compounds and nitrophenyl-glucoside, to represent these classes. PAED showed superior detection limits over low wavelength UV detection for nitroglycerin (PAED = 0.3ppb, UV at 220nm = 48ppb), demonstrating PAED’s applicability to determining nitro-pharmaceuticals. Conversely, UV detection at 220nm proved to be more sensitive than PAED for nitrophenyl-glucoside (UV at 220 = 0.6ppb, PAED = 3.6ppb). However, when nitrophenyl-glucoside was spiked into urine, PAED determination resulted in 99+0.3% recovery, while UV at 220nm resulted in 116+0.2% recovery, suggesting that UV determination may suffer from matrix interference.

A series of in-line curvature sensors on a garment are used to monitor the thoracic and abdominal movements of a human during respiration. These results are used to obtain volumetric tidal changes of the human torso showing reasonable agreement with a spirometer used simultaneously to record the volume at the mouth during breathing. The curvature sensors are based upon long period gratings written in a progressive three layered fibre that are insensitive to refractive index changes. The sensor platform consists of the long period grating laid upon a carbon fibre ribbon, which is encapsulated in a low temperature curing silicone rubber. An array of sensors is also used to reconstruct the shape changes of a resuscitation manikin during simulated respiration. The data for reconstruction is obtained by two methods of multiplexing and interrogation: firstly using the transmission spectral profile of the LPG's attenuation bands measured using an optical spectrum analyser; secondly using a derivative spectroscopy technique.

An optical temporal log-slope difference mapping approach is proposed for cancerous tumor detection; in which target tissues are illuminated by near-infrared ultrashort laser pulses, and backscattered time-resolved light signals are collected. By analyzing the log-slopes of the temporally decaying signals, a log-slope distribution on the detection surface is obtained. After administration of absorption contrast agents, the presence of cancerous tumors increases the decaying steepness of the transient signals. The mapping of log-slope difference between native tissue and absorption-enhanced cancerous tissue indicates the location and projection of tumors on the detection surface. In this paper, we examine this method in the detection of tumor inside a model tissue through Monte Carlo simulation. The tissue model has a spherical tumor of different sizes embedded at the tissue center. It is found that tumors with size not less than 4 mm in diameter in the tissue model can be accurately projected on the detection surface by the proposed log-slope difference mapping method. The image processing is very fast and does not require any inverse optimization in image reconstruction. Parametric studies are conducted to examine to the influences of absorption contrast, tumor size and depth.

In this paper we describe the development of a novel fiber optic probe for subsurface tumor diagnostics, based on non-resonant multiphoton photoacoustic spectroscopy (NMPPAS). In this technique, endogenous biomarkers present in tissues are irradiated in the near infrared, using a tunable high-power laser. The resulting multiphoton excitation events are detected as an acoustic (i.e. ultrasonic) signal, using an ultrasonic piezoelectric transducer. The signal from the piezoelectric transducer is then corrected for laser power fluctuations by normalizing the NMPPAS signal at each wavelength with the laser intensity recorded, from an optical diode. By scanning the laser excitation over the appropriate wavelength range for the tissue of interest, absorption differences between normal and tumor tissues can be measured and analyzed. The fiber optic probe was characterized and optimized for transmission efficiency as well as its time dependent response to high power laser pulses. The focusing optics were optimized and a piezoelectric transducer film detector chosen based on its sensitivity in the ultrasonic frequency range of interest. Using this probe system NMPPAS measurements were performed on several common fluorescent dyes including rhodamine 6G as well as well-characterized biomarkers like tryptophan. Furthermore, the technique was further successfully applied to the differentiation of tumorous and healthy human brain tissues.

The development of small surface plasmon resonance (SPR) sensors to detect biological markers for myocardial ischemia (MI), spinal muscular atrophy (SMA), and wound healing was achieved at low ng/mL and in less than 10 minutes. The markers of interest for MIs are myoglobin (MG) and cardiac Troponin I (cTnI). The limits of detection for these markers are respectively 600 pg/mL and 1.4 ng/mL in saline solution. To study SMA, the level of survival motor neuron protein (SMN) was investigated. A limit of detection of 990 pg/mL was achieved for the detection of SMN. The interactions of SMN with MG decreased the signal for both SMN and MG. Interleukin 6 and tumor necrosis factor alpha (TNFa) were investigated to monitor wound healing. The sensor's performance in more complex solutions, e.g.: serum, showed a large non-specific signal. Modifying the support on which the antibodies are attached improved the sensor's stability in serum by a factor of 5. To achieve this non-specific binding (NSB) reduction, different polysaccharides, biocompatible polymers and short chain thiols were investigated.

Grip strength is an easy measure of skeletal muscle function as well as a powerful predictor of disability, morbidity and mortality. In order to measure the grip strength, a novel fiber optic approach is proposed and demonstrated. Strain dependent wavelength response of fiber Bragg gratings (FBGs) has been utilized to obtain the strength of individual fingers. Five FBGs are written at different center wavelengths on a single photosensitive fiber. Each FBG is used to get the response from each individual finger. The fiber containing the gratings is attached to a suitable grip holder, which can effectively transfer the grip force to the FBGs. An additional reference FBG is also provided to make the device temperature insensitive. Experimental results show that the wavelength shifts of the order of 0.2-0.5 nm can be achieved for individual fingers. The device is calibrated in terms of load to convert the wavelength shift to the strength of the grip. The time dependent wavelength fluctuations was also studied and presented in this paper.

Recently, polarization based optical approaches have received considerable interest due to their potential medical applications. Glucose, a chiral molecule, has the ability to rotate the plane of linearly polarized light, commonly referred to as optical activity, as well as affecting the refractive index of the media which is therefore affects the overall scattering coefficient in a given media. The magnitude of each effect is related to the concentration of glucose. Based on these effects, it would be expected that a change in glucose concentration would alter the diffuse reflectance polarization patterns from turbid media. In this study, we investigate how each of these effects is correlated to glucose concentration in a physiological range for highly scattering biological media. Furthermore, it is shown how diffusely polarized imaging when coupled with chemometrics techniques can be used to quantify glucose concentration.

The fabrication parameters necessary for the development of waveguides that transmit energy from deep ultraviolet to infrared range on wide band gap semiconductor thin film is discussed. Such waveguides in conjunction with microfluidic systems may be used for a spatial and temporal drug delivery in neural tissue. These waveguides may also be suitably modified and employed for novel applications like lab-on-a-chip technologies for Raman Spectroscopy and high speed telecommunication optical switches. Highly textured AlN thin films are grown on C-plane sapphire with high refractive index buffer layer by plasma source molecular beam epitaxy (PSMBE). Analytical measurements such as atomic force microscopy (AFM), ultraviolet spectroscopy and X-ray diffraction, were used to characterize surface morphology and crystalline structure of these films. The fabrication of waveguide structures was performed using laser micromachining with a KrF Excimer laser of wavelength 248 nm and pulse duration of 25ns. Waveguide etching rate for the AlN thin films is investigated as a function laser pulse energy and number of pulses. It is found that etching rate increases almost linearly with both--the pulse energy and number of pulses.

Due to the importance of fluoride in clinical treatment of osteoporosis and its toxicity from over accumulation in bones there is an increased interest in developing selective optical methods for the detection of fluoride anion. Anion recognition and sensing are of interest because of their importance in biological environmental assays and efforts are paid for developing sensitive methods. We synthesized salicylidene furfurylamine 1 and studied spectral properties. Compound 1 fluoresced strongly and the fluorescence was strongly enhanced in the presence of anions as fluoride at low concentrations. A substantially red-shifted emission in acetonitrile was observed. The excitation at 390 nm and the emission was observed at 469nm. Fluoride showed strong absorption and fluorescence enhancement with a significant Stokes shift. Acetate, dihydrogen phosphate, showed small effect, while chloride, bromide had no significant effect.

Near infrared spectroscopy as a neuroimaging modality is a recent development. Near infrared neuroimagers are typically safe, portable, relatively affordable and non-invasive. The ease of sensor setup and non-intrusiveness make functional near infrared (fNIR) imaging an ideal candidate for monitoring human cortical function in a wide range of real world situations. However optical signals are susceptible to motion-artifacts, hindering the application of fNIR in studies where subject mobility cannot be controlled. In this paper, we present a filtering framework for motion-artifact cancellation to facilitate the deployment of fNIR imaging in real-world scenarios. We simulate a generic field environment by having subjects walk on a treadmill while performing a cognitive task and demonstrate that measurements can be effectively cleaned of motion-artifacts.