We have developed a new fluorescence-based biosensor for sensitive detection of species involved in a multivalent interaction. The biosensor system utilizes specific interactions between proteins and cell surface receptors, which trigger a receptor aggregation process. Distance- dependent fluorescence self-quenching and resonant fluorescence energy transfer mechanisms were coupled with a multivalent interaction to probe the receptor aggregation process, providing a sensitive and specific signal transduction method for such a binding event. The fluorescence change induced by the aggregation process can be monitored by different instrument platforms, e.g. fluorimetry and flow cytometry. In this article, a sensitive detection of pentavalent cholera toxin which recognizes ganglioside GMI has been demonstrated through the resonant energy transfer scheme, which can achieve a double color change simultaneously. A detection sensitivity as high as 10 pM has been achieved within a few minutes (c.a. 5 minutes). The simultaneous double color change (an increase of acceptor fluorescence and a decrease of donor fluorescence intensity) of two similar fluorescent probes provides particularly high detection reliability owing to the fact that they act as each other's internal reference. Any external perturbation such as environmental temperature change causes no significant change in signal generation. Besides the application for biological sensing, the method also provides a useful tool for investigation of kinetics and thermodynamics of a multivalent interaction.

Porous silicate materials made by low temperature sol-gel process are promising host matrices for encapsulation of biomolecules. Their mechanical strength, chemical inertness, hydrophilic nature, and above all, their optical transparency makes them an exciting platform for development of biosensors. To date, researchers have focused on sol-gel routes using alkoxides for encapsulation of biomolecules. However, formation of alcohol as a byproduct is an undesired complication as it can have detrimental effect on the activity of entrapped biomolecules. We have developed a novel sol-gel process to encapsulate biological molecules (such as enzymes, antibodies and cells) that uses neutral pH, room temperature, and does not generate alcohol as a byproduct. The process uses sodium silicate as precursor and is carried out in two steps--preparation of a low pH silicate sol followed by gelation at neutral pH in a buffer containing biomolecules. We developed a novel homogeneous immunoassay for 2,4,6-trinitrotoluene (TNT), and have encapsulated the immunoassay reagents in sol-gel matrices to product dispersible biosensors for the detection of TNT. Using the sol-gel doped with immunoassay reagents, we can detect TNT at low ppm levels. We also report encapsulation of E. Coli cells expressing the enzyme organophosphorus hydrolase on the cell surface in sol-gel matrices. The cell- doped sol-gel material can be used to develop biosensors for detection of organophosphates.

The AMBRI Ion Channel Switch biosensor is a novel scanning technology with broad application in a variety of fields. The technology is based on an artificial bilayer membrane attached to gold through hydrophilic tethers. The lamellar bilayer membrane possesses electrical characteristics similar to black (bilayer) lipid membranes being sealed with a capacitance of approximately 0.6 (mu) F/cm², is fluid, and is stable to a variety of media including plasma and whole blood and to challenges with solvent solutions. Receptors/antibodies can be attached to the membrane through biotin-streptavidin linkages, and use of caged biotin species allows optical patterning of the membrane surface.

Vibrational spectra of biomimetic membranes have been obtained using a broad-band approach to sum frequency generation (SFG). A new innovation, broad band SFG (BBSFG) allows for high quality SFG spectra with rapid collection times. With the BBSFG approach, we have followed in situ the formation of a hybrid bilayer membrane from the reorganization of phospholipid vesicles at akanethiol monolayers.

The fluorescence of an ionic conjugated polymer can be efficiently quenched by the small molecular quenchers wherein one quencher molecule may quench the fluorescence of virtually an entire polymer chain. This leads to `static' Stern-Volmer quenching constants as high as 10⁷ - 10⁹ M^-1. A new class of highly sensitive chemical and biological sensors has been developed based on this unique quenching phenomenon.

Planar optical waveguides may be used for the sensitive real-time interrogation of the optical properties of very thin films attached at surfaces, and microfabrication allows the definition of integrated optical circuits, including electrodes, for sensor arrays. Sensing techniques based upon thin-film absorption and refractive index changes are presented, including electrochemically-controlled surface reactions, and present work on fluoroimmunosensor arrays and instrumentation are discussed. In particular, integrated optical immunoprobes for environmental analysis are described.

We have developed a method for simple and highly sensitive detection of multivalent proteins using an optical waveguide sensor. The optical biosensor is based on optically tagged glycolipid receptors imbedded within a fluid phospholipid bilayer membrane formed on the surface of a planar optical waveguide. The binding of multivalent toxin initiates a fluorescence resonance energy transfer resulting in a distinctive spectral signature that is monitored by measuring emitted luminescence above the waveguide surface. The sensor methodology is highly sensitive and specific, and requires no additional reagents or washing steps. Demonstration of the utility of protein-receptor recognition using planar optical waveguides is shown here by the detection of cholera toxin.

In this paper, different luminescence-based sensing configurations and examples of applications in bioaffinity assays relevant for the key life science areas, demonstrating the achieved system performance, will be presented and compared with literature results from refraction-based techniques.

A new optical technique is described that permits extension of cavity ring-down spectroscopy (CRDS) to surfaces, films, and liquids. As in conventional CRDS, the photon intensity decay time in a low loss optical cavity is utilized to probe optical absorption. Extension to condensed matter is achieved by employing intra-cavity total internal reflection (TIR) to generate an evanescent wave that is especially well suited for thin film chemical sensing. Tow general monolithic cavity designs are discussed: (1) a broadband, TIR-ring cavity that employs photon tunneling to excite and monitor cavity modes, and (2) a narrow bandwidth cavity that utilizes a combination of TIR and highly reflective coatings. Following a qualitative description of design features, a beam transfer matrix analysis is given which yields stability criteria and mode properties as a function of cavity length and mirror radius of curvature. A signal- to-noise ratio calculation is given to demonstrate the evaluation of sensitivity.

Surface plasmon resonance spectroscopy is becoming an increasingly important technique in biotechnology and chemical sensing. We present two simple, low cost, high sensitivity devices. The first is laser based mechanical implementation of a Kretschmann setup. Angle sweep is realized in two stages: step motor is used for coarse angle setting, and continuous angle sweep is achieved with a mirror on a floppy disk drive and a cylindrical lens setup. A single detector with AD converter defines resolution of the device through its sampling speed and dynamic range, so high sensitivity can be achieved. Sensor probes are metal- coated microscope slides and sample volume is temperature controlled. Second devices is a disposable cuvette for use in a VIS spectrometer. Specially designed monolithic polycarbonate block provides the required optical path and appropriate incidence angle on a thin metal film, deposited on the block. No equipment is necessary and the cuvette can be used within special cell, such as temperature controlled vessel. Device is also discussed in view of a low cost fiberoptic implementation. Some experimental results are presented to prove the applicability of devices. Disadvantages of technical solutions, used in devices, are also taken into consideration.

The need for small, fast responding detection systems is growing and fiber-optic bead arrays offer a different approach to small sensor design. Sensor arrays are fabricated by inserting self-encoded microspheres into microwells etched into the distal face of an imaging fiber. Each imaging fiber is 0.5 - 1 mm in outer diameter and consists of 5,000 - 10,000 individually clad, 3 - 4 micrometers diameter optical fibers bundled together. The bundles are coherent, allowing each microsphere in a well to be addressed as an individual sensor. Microsphere sensors are silica or polymer beads (approximately 3 micrometers in diameter) impregnated with solvatochromic dyes. These dyes alter their fluorescence emission spectra in response to changes in vapor polarity, allowing analytes to be discriminated based on their signature fluorescence response over time. A computational network is trained to recognize these response patterns for each sensor type, allowing for identification of specific organic vapors. Each sensor type is cross- reactive, and has unique fluorescence response patterns to different analytes. The sensor types can be identified based on their unique responses, allowing their position to be registered by observing the identity of the response pattern toward a known standard. Such encoding enables array fabrication to be simplified since sensors can be randomly dispersed throughout the array, instead of specifically patterned within the array. Possible applications for bead array detectors include environmental and industrial monitoring, land mine detection, and medical diagnostics.

In this paper, we investigate the use of fluorescently- labeled binding proteins and genetically engineered bacterial cells for sensing of phosphate, glucose, and L- arabinose. To optimize the performance of the labeled binding proteins for biosensing purposes, a few key considerations were taken into account. A site-selective labeling protocol of the fluorescent reporter to the protein was used to ensure that the probe reported from a specific domain of the protein. The labeling sites chosen were hypothesized to undergo a physicochemical change when the biorecognition element binds the analyte. Cysteine mutations were introduced into the binding proteins by site-directed mutagenesis using the polymerase chain reaction. The residues selected were all in close proximity to the binding cleft, a region that is affected the most by the conformational change that accompanies ligand binding. The cysteine residues were then labeled with environment- sensitive fluorophores and changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. The application of microorganisms in sensing systems represent new advances in the development of novel analytical techniques for the detection of a target analyte. In these systems, a genetically engineered organism generates an analytically useful signal when it encounters a specific target substance due to selective recognition and binding properties towards that particular compound. This concept has been demonstrated using an optical bacteria-based sensing system capable of detecting the monosaccharide L-arabinose that employed the green fluorescent protein as a reporter protein.

Advances in laser devices, control electronics, and data processing software have enabled significant improvements in tunable diode laser sensor design in the last several years. Miniaturization of the primary subsystems and development of robust data analysis software for space applications has enabled diode laser sensors to be considered for applications where they previously were not feasible due to size, weight, power constraints, or cost. The substantial reductions in diode laser sensor footprint and power consumption achieved at the Jet Propulsion Laboratory between 1992 and 1996 have led to the development of hand- held, battery-operated sensors for the Space Station, the fist space-qualified diode laser sensors presently en route to Mars, and a small humidity sensor suitable for use on commercial aircraft. An overview of the current status of sensor development at SpectraSensors and JPL is presented in this paper.

Diode lasers emitting in the wavelength range between 1.7 and 4 micrometers are of particular interest for a large number of applications such as remote gas sensing, molecular spectroscopy and medical applications. Distributed feedback lasers show a singlemode emission and are therefore ideally suited for sensing applications. However the fabrication of DFB lasers usually comprises an overgrowth step of etched gratings, which critically determines the laser properties. We have developed high quality GaInAsSb/AlGaAsSb laser layers and have realized DFB lasers from them without the need to develop overgrowth processes. The laser structures were grown by solid source molecular beam epitaxy on n-GaSb substrates. Depending on the In and the As incorporation in the quantum well the emission wavelength can be varied from 1.5 up to 2.1 micrometers . Near 2 micrometers excellent room temperature performance has been obtained for strained Fabry Perot quantum-well lasers. Ridge waveguide lasers were realized by photolithography and reactive-ion etching. Using e-beam lithography and a Cr-metal lift off, DFB gratings were deposited on each side of the ridge. By using an appropriate grating period an emission wavelength around 2 micrometers was realized. The lasers have clean single mode spectra, threshold currents of less than 40 mA and deliver output powers in excess of 5 mW per facet.

Combinatorial methods have been used to generate nucleic acid molecules with specific characteristics. Aptamers are nucleic acid binding species, and can be modified to directly transduce molecular recognition to optical signals. Aptazymes are allosteric or effector-activated ribyzymes. We have designed or selected aptazymes that are responsive to a variety of ligands. In particular, we have selected a ribozyme ligase that is activated 10,000-fold in the presence of an oligonucleotide effector, and have designed ligases that are up to 1,600-fold dependent on small molecule effectors. Even in those instances where designed constructs were initially unresponsive, we have been able to use selection to optimize their response characteristics.

Naturally occurring motifs have been redesigned to product fluorescent peptidyl-chemosensors that sensitively and selectively recognize Cu(II) or Fe(III). The modular nature of peptide architecture allows preparation and evaluation of potential sensors on solid supports.

The development of molecular devices based on immobilized protein films is currently a very active area of research worldwide. Detailed structural and functional characterization of these films is a prerequisite to the rational development of deposition methods that product bioactive structures, but is a technically difficult challenge. Two recent thrusts in our work have been investigated of methods designed to create macroscopically ordered arrays of protein molecules, and development of new optical techniques to characterize the ensemble properties of these arrays.

A phase-sensitive cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique features for making frequency-domain lifetime measurements on free fluorophore (solution) and on fluorophore-labeled cells/particles in real time. No other instrument can quantify lifetimes directly and resolve heterogeneous fluorescence based on differences in lifetimes (expressed as phase shifts), while maintaining the capability to make conventional flow cytometric measurements. The technology has been characterized with respect to measurement precision, linearity, sensitivity, and dynamic range. Fluorescence lifetime distributions have been measured on autofluorescence lung cells, thymocytes labeled with antibody conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, cells stained with DNA-binding fluorochromes, and on particles labeled with fluorophores and free fluorophore (solution). Phase-resolved, fluorescence signal- intensity histograms have been recorded on thymocytes labeled with a phycoerythrin/Texas Red tandem conjugate and propidium iodide to demonstrate the resolution of signals from highly overlapping emission spectra. This technology adds a new dimension to flow analyses of free and cell/particle-bound fluorophore. Lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

The response time of biosensors which reversibly bind an analyte such as a metal ion is necessarily limited by the kinetics with which the biosensor transducer binds the analyte. In the case of the carbonic anhydrase-based biosensor we have developed the binding kinetics are rather slow, with the wild type human enzyme exhibiting an association rate constant ten thousand-fold slower than diffusion-controlled. By designed and combinatorial means the transducer may be mutagenized to achieve nearly diffusion-controlled association rate constants, with commensurate improvement in response. In addition, a variant of apocarbonic anhydrase has been immobilized on quartz, and is shown to response rapidly to changes in free copper ion in the picomolar range.

Water-general-purpose solvent and a important component of uncountable set of objects of the living and lifeless nature. The development of molecular and structural-chemical representations has allowed to give depleting explanation of a special talent of molecules of water to derivate connection with fragments almost of any matters. Began to be elucidated also role of `bonding' water in originating major physical characteristics of hydrated matters--clay, gypsum, of a cement rock some types of ferroelectric. At last, after discovery of L. Poling in 1961 of intercoupling between a phenomenon of a narcosis and crystallization of hydrates of drug matters became apparent, that the water, bound with biological molecules, somehow participates in control of biochemical processes.

An optical fiber based low coherence interferometer for measuring the Epithelium thickness of Bronchial tissue, for early diagnosis of Carcinoma in situ, is presented. Previous simulation of laser induced fluorescence using an electromagnetic scattering model has extracted the relative permittivity value for the Submucosa and Epithelium layers indicating a difference of up to 0.14. The optical system presented here uses a low coherence source operating at 840 nm with a bandwidth of 30 nm, coupled into single mode optical fiber. A Fizeau cavity is formed between the fiber end and the tissue under investigation. A remote processing interferometer is used to monitor changes in permittivity between the different tissue layers. An initial experiment has demonstrated a sensitivity measurement of 40 dB for a permittivity difference measurement of 0.61. Preliminary results have shown that the discontinuity between the Bronchial Epithelium layer and its surrounding medium can be identified allowing the thickness of the Epithelium layer to be measured to an accuracy of 20 micrometers . Since interferometric noise contributions are only significant within the processing interferometer, the fiber optic Fizeau interferometer technique is a strong candidate for the development of an endoscope for the early detection of cancer within Gastrointestinal and Respiratory tracts.

Possibility of localized doping by Er³⁺ diffusion at moderate (less than 500 degree(s)C) temperature is demonstrated for lithium niobate and sapphire. The doping is achieved by immersing the substrate wafers into reaction melt containing small amounts of erbium salt. A crucial point of the presented technology is a crystallographic orientation of the used wafers. Though in the Z- and Y-cuts of lithium niobate the content of incorporated erbium did not exceed the concentration achieved using standard high temperature (or high energy) approaches, lithium niobate X-cuts contained up to 10 weight % of erbium. Similar results were obtained also for the corresponding cuts of sapphire. This strong anisotropy of the doping is explained on the basis of crystal structure of the particular cuts. The in-diffused layers in all the cuts are rather shallow, however, erbium can be diffused deeper into the substrates by post-diffusion annealing in air. The moderate-temperature approach enabled us to fabricate APE waveguides in erbium doped lithium niobate without deteriorating the samples surfaces. The samples were characterized by RBS, SEM, NDP and mode spectroscopy.

We present results of our study of concentration profiles of lithium (c_Li) in annealed proton exchanged (APE) waveguiding layers as measured by the neutron depth profiling method. This non-destructive method is based on the ⁶Li(n,(alpha) )³H reaction induced by thermal neutrons and allowed easy monitoring of c_Li profiles in a large number of samples fabricated under various fabrication conditions. Our systematic study revealed that there was no linear relationship which unambiguously attributed (Delta) n_e to (Delta) c_Li, on contrast with up to now generally accepted opinion. Every particular waveguide has very similar mirror-shaped n_e as well as c_Li depth profiles, but, generally, all the waveguides can not be characterized with the same n_e vs. c_Li relationship. The most important fabrication step has appeared to be the post-exchange annealing, during which lithium atoms are transported towards the sample surfaces. The annealing regime pre-destined not only the depth distribution of lithium atoms, but as a consequence of it, also other properties of the waveguiding region. We have formulated n_e vs. c_Li semi-empirical relationship and listed a set of case-dependent empirical constants for our fabrication system. That allows us to fabricate the APE waveguides with a priori given properties for a wide range of special applications.

A novel synthetic peptide spacer designed on a gold film is introduced for use in surface plasmon resonance (SPR) sensing. The peptide was a specially designed sequence of amino acids, synthesized by the Fmoc-solid-phase chemistry. The peptide was adsorbed on the gold film from an aqueous solution via its four thiol groups, forming a self-assembled negatively charged monolayer. The monolayer contained carboxyl groups, which were activated by the EDC/NHS technique. It was successfully used as a matrix (2 - 4 nm thick) for covalent immobilization of fusion protein, which included C-terminal fragment of human 5-hydroxytryptamine transporter (molecular weight approximately 21 kDa). The reaction between the immobilized protein and antibodies was monitored by SPR means. The matrix did not cause degradation of immobilized components and steric hindrances to mass transport, and also demonstrated low nonspecific binding to antibodies. Besides, the matrix could be regenerated without decreasing SPR response to the reaction. Along with the ability to immobilize high weight molecules, which are unable to enter a conventional CM-dextran matrix due to steric hindrances to mass transport, the peptide matrix has a number of advantages over the CM-dextran matrix, namely, simplicity in preparation, low cost, and much shorter time needed for preparation. The peptide spacer matrix can be widely used not only in SPR, but also in other analytical techniques that require immobilization of proteins on metal surfaces, such as interferometry, piezoelectric detection, scanning tunneling microscopy.

Thin carbon or carbon nitride films exhibit specific optical and mechanical properties which make them promising materials for integrated optical sensors. Carbon and carbon nitride thin films were prepared by the Plasma Enhanced Chemical Vapor Deposition (PECVD) method. The substrates were prepared in shapes and sizes suitable for the planned measurements. The surfaces for film deposition were polished and the substrates were cleaned in organic solvents by means of ultrasound before treatment. The following film properties were investigated: the thickness, internal stress, dependence of the friction coefficient on load, refractive index, waveguide properties and composition. The most interesting feature of these films is a change of their refractive index under the influence of the surrounding gaseous environment. Carbon nitride films were prepared in a PECVD apparatus in an arrangement with planar plate electrodes through the decomposition of methane in argon atmosphere. CN_x films were prepared by the reaction of methane and nitrogen or methane and ammonia. It has been found that optical and mechanical properties of the films prepared on the positive and negative electrodes are greatly different. The films deposited on the negative electrode were harder and their refractive index was higher than those deposited on the negative electrode. The refractive index of the harder films ranged from 2.2 to 2.6 while for the softer films is ranged from 1.6 to 1.8. Using ellipsometry it has also been found that the refractive index of the films prepared on the positive electrode did not change under the atmosphere of H₂. On the other hand, the refractive index of the films deposited on the negative electrode and measured under the same conditions changed by about 3 - 5%.

Nonlinear optical systems are presented, giving possibility to change complex light fields characteristics with the aim of image processing on the base of nonlinear optical effects, such as stimulated scatterings of light, two-photon excited luminescence, Kerr-effect etc. These systems allow to perform image processing, changing characteristics of the complex light field, forming the image. For instance, we can change image contrast from that of initial image to the contrast inversion. It's possible also to visualize phase and low-absorbing objects, phase inhomogeneities in transparent materials, thickness variations of low-absorbing layers and roughness of reflecting surfaces. Thus, these methods can find wide applications in materials technologies monitoring, biology and medicine. Image processing is performed in real-time scale, which permits to avoid influence of effects, connected with light beam propagating in materials, and to study quick processes.

The model of the pulse laser nephelometry polarization methods of using in biotechnologies and ecology is elaborated. It is shown that informativity of the laser nephelometry measurements increases when polarization self- action effects of short pulses of the laser radiation are taken into account by multiple scattering is dispersed biological media with high concentration of weighted particles. The soliton and simulton regimes of the polarization transformation of the laser pulses have been investigated. Conditions of the modulation instability are determined of the pulse laser radiation of arbitrary polarization in nonlinear scattering biomedia.

We demonstrated the results of investigations on the creation of the new high-precision laser methods and technologies of the on-line detection and operative quantitative analysis of the small and super-small concentrations of sugar, glucose, fructose and other matters dissolved in the transparent liquids based upon the effect of the natural optical activity of such matters. The record- breaking improvement has been achieved of the sensitivity of the registration systems as well as measurement precision in determination of small rotation angles of the radiation polarization plane due to the natural optical activity of solutions.

We have developed an instrument that measures fast transient birefringence and polymer chain orientation angle in complex fluids. This instrument uses a dual-crystal transverse Electro-Optic modulator with second crystal's modulation voltage applied 180 degree(s) out of phase from that of the first crystal. The precise measurement of birefringence using this instrument strictly depends on three separate signals. These are the dc, first and second harmonic voltages. The harmonics are relatively noise-free while the dc signal is subject to appreciable noise. Therefore, wavelet based noise reduction techniques have been used to denoise the measured dc signal. The de-noised signal was recombined with the harmonic signals to obtain retardation and birefringence measurements using a high precision 1/16 wave plate in a rotating mount to simulate a birefringence sample.

The optical properties and nonlinear response of photorefractive crystals of quartz and barium sodium niobate (BSN) are investigated in the temperature range of 20 - 700 degree(s)C. The specl-structure of the laser beam in quartz crystal was observed in the narrow range of temperature at (alpha) -(beta) phase transition. The elastic light scattering intensity of the quartz sharp increases at phase transition and drops after. The specl-structure in BSN crystal appears in the wide range of the temperature, from room temperature to the temperature of ferroelectric phase transition, Tc equals 560 degree(s)C. The elastic light scattering in the BSN crystal rapidly decreases at the temperature Curie. The change of intensity of the elastic light scattering corresponds to the visually observed change in the laser beam trace in the crystals. The anomalies of scattering light probably reflect the structural changes associated with the formation of incommensurate phase in these crystals.