A new glucose sensing system based on near infra-red fluorescence resonance energy transfer (FRET) from CocanavalinA-allophycocyanin to dextran labelled malachite green is demonstrated. Single-photon timing fluorescence lifetime measurements have enabled us to investigate and understand the quenching kinetics in terms of the dimensionality of energy transfer.

Assays which discriminate and enumerate dying or dead cells are important in various types of cellular studies. In many instances, there is a need to identify dead cells that interfere with fluorescent probes which are used to measure functional and physiological properties in viable cells. For example, dead cells can introduce analytical errors arising from (1) nonspecific uptake of fluorescent probes, leading to erroneous percentages of positive labeled cells, (2) increased autofluorescence, and (3) altered antigen expression. The ability to detect dead cells is also of importance in determining the effectiveness of cytotoxic agents. Propidium iodide (PPI) exclusion, which is analogous to the non- fluorescent trypan blue dye test for viability, is used extensively in flow cytometry assays. However, the use of PI can potentially limit the application of additional fluorescent probes due to spectral overlap of the probe with PI. In this report we present phase-resolved fluorescence studies on rat and murine thymus cells labeled with phycoerythrin-antiThy 1.1 and phycoerythrin/Texas Red-antiThy 1.2 immunofluorescence markers, respectively, and PI. Overlapping emission spectra are resolved based on differences in fluorescence lifetimes of the probes and PI. These studies demonstrate a new lifetime-based viability method for use in analysis of immunofluorescent probes and for assaying the dynamics of cell killing.

Most assays for drug screening are monitoring the metabolism of cells by detecting the NADH content, which symbolize its metabolic activity, indirectly. Nowadays, the performance of a LASER enables us to monitor the metabolic state of mammalian cells directly and on-line by using time-resolved autofluorescence detection. Therefore, we developed in combination with tissue engineering, an assay for monitoring minor toxic effects of volatile organic compounds (VOC), which are accused of inducing Sick Building Syndrome (SBS). Furthermore, we used the Laserfluoroscope (LF) for pharmacological studies on human bone marrow in vitro with special interest in chemotherapy simulation. In cancer research and therapy, the effect of chemostatica in vitro in the so-called oncobiogram is being tested; up to now without great success. However, it showed among other things that tissue structure plays a vital role. Consequently, we succeeded in simulating a chemotherapy in vitro on human bone marrow. Furthermore, after tumor ektomy we were able to distinguish between tumoric and its surrounding healthy tissue by using the LF. With its sensitive detection of metabolic changes in tissues the LF enables a wide range of applications in biotechnology, e.g. for quality control in artificial organ engineering or biocompatability testing.

Delayed luminescence (D.L.) is a measure that provides important information on biological systems fields, structures and activities, by counting impinging and emitted photons. Many recent experimental works have shown the existence of a close connection, sometimes analytically expressed between the biological state of the system and D.L. parameters. Our investigations aim to show that D.L. is a workable analytical technique covering a large number of disciplinary fields, from agriculture to pollution control and from medical diagnostics to food quality control. The authors have conducted systematic research about D.L. from unicellular alga Acetabularia acetabulum to Saccharomyces cerevisiae yeast cultures and about more complex systems such as Soya seed (Glycine max, L.) and its dependence on sample preparation, history, intracellular signaling, metabolism and pollutant presence. We will discuss the most relevant results together with theoretical considerations on the basic interaction at work between biological systems and electromagnetic fields.

We devised an optical assay for glucose based on the genetically-engineered glucose/galactose binding protein (GGBP) from E. coli and phase-modulation fluorometry. A single cysteine mutation was introduced at position 26 of GGBP. When labeled with the sulfhydryl-reactive probe I-ANS, GGBP showed a more than 50% decrease in florescence intensity with increasing glucose concentration (K_d approximately 1 (mu) M). This is consistent with the glucose-bound structure of GGBP where residue 26 becomes more exposed to the aqueous media. Since minimal lifetime changes were observed with glucose binding, a modulation sensor was devised wherein a long lifetime ruthenium metal-ligand complex (Ru) was painted on the surface of the cuvette containing ANS26-GGBP. Glucose binding resulted in changes in the relative intensities of ANS26-GGBP and Ru which were observed as dramatic changes in the modulation at a low frequency of 2.1 MHz. The modulation measured at 2.1 MHz accurately determines the glucose concentration to plus or minus 0.2 (mu) M.

A series of chiral macrocyclic lanthanide complexes has been devised whose metal-based luminescence (Eu, Tb or Yb) is a function of a defined variable or set of variables. Complexes have been devised in which the lifetime or intensity of emission is a sensitive function of pH, pO₂, or halide ion concentration. The pH dependent systems report either the excited state singlet pK_a (Eu) or the triplet pK_a of an integral phenanthridinium chromophore, with luminescence enhancements of over 500. The halide and oxygen dependent systems operate differently via a quenching of the intermediate excited aryl singlet and triplet states respectively. In addition, complexes have been devised in which reversible binding of hydrogencarbonate displaces two bound water molecules, leading to an enhancement of the metal- based emission intensity (Eu, (Delta) J equals 2) or lifetime. At the same time, pronounced changes in the polarization of emission occur, signalling changes in the helicity at the metal center. Hydrogencarbonate chelates preferentially in the concentration range 20 to 40 mM in the presence of phosphate, lactate, citrate and halide at physiological levels.

Sensor systems have long been needed for detecting the presence in solution of certain chemically or biologically important species. Sensors are used in a wide range of applications from simple litmus paper that shows a single color change in acidic or basic environments to complex biological assays that use enzymes, antibodies and antigens to display binding events. With this work the use of boronic acids in the design and synthesis of sensors for saccharides (diols) will be presented. The fluorescent sensory systems rely on photoinduced electron transfer (PET) to modulate the observed fluorescence. When saccharides form cyclic boronate esters with boronic acids, the Lewis acidity of the boronic acid is enhanced and therefore the Lewis acid-base interaction between the boronic acid and a neighboring amine is strengthened. The strength of this acid-base interaction modulates the PET from the amine (acting as a quencher) to anthracene (acting as a fluorophore). These compounds show increased fluorescence at neutral pH through suppression of the PET from nitrogen to anthracene on saccharide binding. The general strategy for the development of saccharide selective systems will be discussed. The potential of the boronic acid based systems will be illustrated using the development of glucose and glucosamine selective fluorescent sensors as examples.

Aiming to develop new fluorescent chemosensors for biological and environmental applications, we have designed and synthesized new chemical species able to reversibly bind alkali, earth-alkali, and transition metal ions. For signaling the binding of the target analyte, we have inserted in the structure of the chemosensors different luminophores, such as dioxyxanthone derivatives, dansyl derivatives, ruthenium complexes, and hydroxyquinoline derivatives. In solution, the binding is always signaled by pronounced changes in the photophysical properties of the inserted luminophore such as emission wavelength and intensity, and excited state lifetime. The mechanism for the signal transduction strongly depends on the chosen receptor and luminophore moieties, and has been investigated in detail by means of steady state and time resolved spectroscopy. In all cases, the synthesized chemosensors have proved to be chemically and photochemically stable. Good selectivity and affinity has been obtained with different sensors for K⁺, Mg²⁺, Ba²⁺, Zn²⁺, Ni²⁺ and Cu²⁺, even in physiological pH conditions. Moreover the use of an array of these sensors in optodes could lead to the construction of the so called electronic tongues. All these features make these sensors promising candidates for analytical applications.

We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye Rhod-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37 degrees Celsius, and is stimulated at 8 Hz, with 100 (mu) g of Rhod-2 bolused through the perfusate. After a 25-minute washout period, approximately 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O₂, thus minimizing interference due to changes in tissue oxygenation. Ca²⁺-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33 plus or minus 9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of Rhod-2 through the perfusate, the reflected absorbance between 524 nm (Rhod-2 sensitive) and 589 nm (Rhod- 2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATP_ase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368 plus or minus 68 nM and 654 plus or minus 164 nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.

We present a comparison between two basically different optical detection systems: a confocal epifluorescence microscope, and a new evanescent wave detection system employing a parabolic optical element. In a microscope set-up, fluorescence light is collected within a cone around the optical axis, whereas in the evanescent light detector, fluorescence light is collected mainly at angles larger than the so-called critical angle of total internal reflection. Based on a thorough theoretical modeling of both experimental set-ups, comparison between the two detection systems is made Particularly, the optical detection efficiency is compared. 5

A new improved fluorescence correlation spectroscopy apparatus for determining motional states of particles, in which the scheme of fluorescence correlation spectroscopy with traveling interference fringe excitation (FCSTFE) is applied, is presented. In this method, the modulated fluorescence signal from particles excited by a moving interference fringe is detected, and cosine and sine Fourier coefficients at the frequency of the traveling fringe are recorded. The autocorrelation function of the fluorescence intensity consists of terms which are characterized by the size of the excitation region and the spacing of the fringe. A lock-in detection method is adopted for extracting cosine and sine Fourier coefficients at the frequency of the traveling fringe [F_cos(t), F_sin(t)]. Autocorrelation functions of F_cos(t) and F_sin(t) express the motion of the fluorescent particles. We constructed the apparatus using acousto-optic modulator (AOM) devices to generate the traveling fringes. The potential of this method for the extraction of information about the dynamics of particles in biological systems is discussed.

Steady-state and time-resolved fluorescence of the single tryptophan residue (W187) of annexin V show that the conformation and the dynamics of domain III are strongly modified upon binding of the protein to negatively charged phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles of water/sodium bis(2- ethylhexyl) sulfosuccinate (AOT) in iso-octane. In the protein at neutral pH, W187 is slightly mobile and buried in a hydrophobic pocket. It becomes more mobile and is moved in a more polar environment when the protein interacts with the model membranes. In each condition, the heterogeneity of the fluorescence intensity decay of W187 is likely due to the co- existence of local conformers with different dynamics. A similar change of conformation and dynamics can be provoked by mild acidic pH. This suggests that electrostatic interactions are important for the folding of domain III. An interplay of salt bridges implying charged amino-acid side-chains at the protein surface in domain III can be observed in the crystal structure. Local pH modifications at the membrane surface can therefore be responsible for the observed conformational change. The high flexibility of domain III in the membrane- bound protein suggests moreover that this domain may not be crucial for the interaction of the protein with the membrane, in agreement with recent atomic force microscope results (Reviakine et al., 1998, J. Struct. Biol. 121, 356-362).

Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

The planar integrated optical waveguide (IOW) is an inherently sensitive geometry for attenuated total reflection (ATR) spectroscopy of interfacial samples. A major disadvantage that has limited its wider use is the difficulty of measuring broadband spectra. Due to the quantized nature of light propagation in a planar IOW, conventional grating and prism couplers are efficient only over a narrow (less than 5 nm) spectral range at a given launch angle. We have developed a multichannel spectrometer capable of measuring a broadband visible ATR spectrum at the surface of a single mode, planar waveguide. The bandwidth is greater than 150 nm, which makes it possible to measure spectra of very weakly absorbing molecular films. We have also developed an electrochemically- active, planar IOW (EA-IOW) that combines the information content of spectroelectrochemistry with the sensitivity of the single mode planar waveguide geometry. An evaluation of this device has demonstrated that highly sensitive spectroelectrochemistry of surface confined films can be performed; the estimated pathlength enhancement is ca. 4,000 relative to a transmission geometry.

Surface immobilizable molecular beacons have been developed for DNA hybridization studies on a silica glass plate. Molecular beacons are a new class of oligonucleotide probes that have a loop-and-stem structure with a fluorophore and a quencher attached to the two ends of the stem. They only emit intense fluorescence when hybridize to their target molecules. This provides an excellent selectivity for the detection of DNA molecules. We have designed biotinylated molecular beacons which can be immobilized onto a solid surface. The molecular beacon is synthesized using DABCYL as the quencher and an optical stable dye, tetramethylrhodamine, as the fluorophore. Mass spectrometry is used to confirm the synthesized molecular beacon. The molecular beacons have been immobilized onto a silica surface through biotin-avidin binding. The surface immobilized molecular beacons have been used for the detection of target DNA with subnanomolar analytical sensitivity. have also immobilized two different molecular beacons on a silica surface in spatially resolved microscopic regions. The hybridization study of these two different molecular beacon probes has shown excellent selectivity for their target sequences. The newly designed molecular beacons are intended for DNA molecular interaction studies at an interface and for the development of ultrasensitive DNA sensors for a variety of applications including disease diagnosis, disease mechanism studies, new drug development, and in the investigation of molecular interactions between DNA molecules and other interesting biomolecules.

In this paper we report the use of phase sensitive fluorometry to obtain preliminary results from opto-chemical fluorescent oxygen nanosensors. PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensors were fabricated by immobilizing tris(4,7-diphenyl-1,10-phenanthroline)Ru(II) chloride and tris(1,10-phenanthroline)Ru(II) chloride within a polyacrylamide matrix. PEBBLEs have diameters of 20 - 200 nm and exhibit excellent performance for dissolved oxygen detection. Their performance is compared with micrometer-sized (10 - 20 micrometer) optical fiber sensors and free dye in solution. Oxygen sensing ability of PEBBLEs was tested in the presence of other quenchers and compared with free dyes in solution. While PEBBLEs have been developed for minimally invasive intracellular chemical analysis they show additional advantages, such as increased dynamic range, compared to microsensors, and an absence of interference (quenching) by heavy ions in contrast to free dye solutions.

We present a novel method to measure Raman spectra from whole bacteria cells by using Surface Enhanced Raman Scattering (SERS). We deposit a silver coat on Escherichia coli and Bacillus megaterium bacteria and measure strongly enhanced (greater than 400,000 fold) and highly reproducible Raman spectra. The spectra are rich but not overly congested, as the surface enhancement is selective to the precise chemical nature of the biochemical molecules, and their proximity to the silver particulate matter. The main bands we observe can be associated with peptides and polysaccharides in the cell- wall and its membrane. The spectra from E. coli (a Gram- negative bacterium) and B. megaterium (a Gram-positive bacterium) are similar in their general form, but differ in detail. The spectrum from a commercial yeast extract is vastly different. This approach can be extended to probe the internal chemical environment within bacteria and applied to the identification of micro-organisms also applied to studying other biochemical problems and phenomena, such as biomineralization, heavy metal toxicity, cell-wall structure and others.

Two intracellular fluorochromes, NAD(P)H and Schiff Bases, provide monitoring of energy metabolism and photoperoxidations. Fluorochrome spectra and topographic distribution are measured in a microspectrofluorometer, pixel by pixel using a CCD. The mitochondrial arrangement of Saccharomyces cerevisie and metabolic activity at nuclear kidney epithelial sites is revealed. A kind of accelerated photoaging results in the accumulation of Schiff pigment. Schiff base emission is red-shifted, and it may be preceded by photo-oxidation of NAD(P)H. UVA production of oxygen radicals and peroxides may influence detoxification, senescence and/or transformation. Besides lysosomes, mitochondrial energy metabolism and ER and Golgi detoxification are open to study as multi-organelle complexes with fluorescent xenobiotics and probes. Melanocytes vs. melanoma cells in culture will be investigated using a new compact interferometer for Fourier coding of both emission and excitation spectra. Surprisingly, the photographic method, using the highest sensitivity films, may sometimes produce excellent structural detail. However, for kinetic studies, the CCD, or equivalent, is required. There is good potential for applications in diagnostics and prognostics plus the evaluation of new biopharmeceuticals.

The choice of metal clusters as signal transducers of molecular binding events is based on their about 1000 times higher extinction coefficients compared to conjugated chromophores. Using cluster based assays it is possible to visualize the binding of biomolecules at a given surface by a bound layer of ligand-modified metal clusters. The success of cluster visualization was mainly based on the significant signal stability contrary to chromophores and especially fluorophores. Cluster probes are not only efficient direct markers but within the past years became the basis of new devices employing cluster resonance, cluster field enhancement, and cluster-cluster interactions. Multilayered highly resonant systems clearly exhibit strong reflection minima induced by the resonant behavior of the metal cluster layer. At least one narrow reflection minimum can be shifted to the red or infra red spectral range and therefore far away from spherical gold colloids to be ready by a 10 micrometer resolution optical scanner type high density device. Even without employing near-field optics spatial resolution is within 100 - 500 nm. The setup enabled us to replace conventional ELISA assays overcoming the various technological limits as there are e.g. multiple incubation steps and spatial resolution. The focus of the development was to provide an optical biochip which allows detection of analytes based on arrays of proteins, DNA and high throughput-screening targets for drug discovery.

The possibility and potential benefits of using an extreme red fluorescent dye such as Cy5.5^TM to label drug discovery target proteins was studied experimentally. Cy5.5 labeled BSA, GFP, and CRP were used as examples of protein-dye conjugates whose binding to corresponding antibody was detected by changes in either rotational or transnational diffusion properties, that is, by either fluorescence polarization or confocal fluorescence correlation spectroscopy (FCS). In addition, FCS was used to quantitate excitation of Cy5.5 to its triplet state. Fluorescence polarization and lifetime were measured as a function of excitation wavelength or glycerol concentration and solvent viscosity.

We describe a new approach to particle size measurement in silica sols based on the decay of fluorescence anisotropy of a bound near infrared dye. Changes in particle size up to 4.5 nm are resolved with sub-nanometer resolution across the sol to gel transition.

We have demonstrated that free metal ions such as Zn(II) can be determined by fuorescence anisotropy (polarization) using an apometalloenzyme, carbonic anhydrase II, and a fluorescent aryl sulfonamide inhibitor of the enzyme whose affinity for the enzyme is metal-dependent. We felt that attaching the fluorescent aryl sulfonamide to the protein would provide a similar response, while avoiding problems of disproportionation of the inhibitor and protein. In fact a tethered aryl sulfonamide ABD-T gave very good results: Zn(II) and Cu(II) at picomolar levels and Co(II), Cd(II), and Ni(II) at nanomolar levels can all be determined by changes in fluorescence intensity, anisotropy, and lifetime using visible excitation sources. Implications of these results are discussed.

We describe a new method for fluorescence sensing based on measurements of the steady state polarization of an analyte- sensitive fluorophore in the presence of a reference fluorophore with known polarization. The basic concept is that the polarization of a mixture reflects a weighted average of the polarization of the emitting species. By use of reference fluorophores the starting values can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the sensing fluorophore due to the analyte result in changes in the polarization of the combined emission. A wide dynamic range is available because of the freedom to select high or low starting polarization values. Polarization-based sensing was demonstrated for pH using 6- carboxy fluorescein. We also show that polarization sensing can be used for measurements of oxygen and glucose. Polarization sensing can have numerous applications in clinical and analytical chemistry.

The baking quality and storage stability of white flour are affected by its non-starch lipids content, and by the proportions of non-polar and polar lipids classes. At present, information on the lipids composition in the various parts of the wheat grain is scarce and their redistribution in the flour millstreams after milling is not well understood. Here we have implemented a novel method based on microspectrofluorometry to investigate lipids distribution in the wheat kernel. This technique has already been a proven tool to study primary fluorescence in wheat grain. For this study Nile Red was introduced as a fluorescent stain to map lipids in different compartments of a wheat transverse section. Microspectrofluorometry allows in situ characterization of lipids material in transverse cut of wheat grain. Florescence spectra were recorded and decomposed into the principal spectral components which can in turn be approximated to the real lipid materials of the wheat. Using these models, spectral fluorescence imaging was performed allowing the spatial organization of lipids in the wheat sections to be obtained.

Donor-Donor Energy Migration (DDEM) and fluorescence anisotropy experiments can be utilized as a versatile tool for examining protein structure and function. For this, pairs of identical fluorescent probes (D) are attached to unique residues created by means of site specific mutagenesis. Present work illustrates the applicability of the method on the latent form of Plasminogen Activator Inhibitor-1 (PAI-1). Different DD-pairs of mutated PAI-1 were prepared and studied, namely; V106C-H185C, H185C-M266C and M266C-V106C. The Cys residues were labelled with a sulfhydryl specific derivative of BODIPY^R [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-yl)methyl iodo-acetamide]. To determine the rate of DDEM within such a pair, intramolecular order and dynamics must be considered (Biophys. J., 74, 11-21, 1997). For analysis of data, additonal information was obtained from experiments with the corresponding D-labelled single Cys mutants, that is, V106C, H185C and M266C. The stability of values determined was tested by generating and re-analyzing synthetic data. The intramolecular distances obtained agree, reasonably well, with those determined from the X-ray structure of latent PAI-1.

Fluorescent molecules, whose spectra or quantum yields are sensitive to their surrounding environments, are valuable in the study of heterogeneous media, organized media and biological media, and many fluorescent solvatochromic dyes have been developed for these applications. However, the existing fluorescent solvatochromic dyes either have short absorption and emission wavelengths, low extinction coefficients, low quantum yields or small Stokes shift. We have recently developed solvatochromic Dapoxyl^TM dyes, whose fluorescence maximum shifts to longer wavelengths, and fluorescence quantum yield decreases with increasing solvent polarity. In these molecules, there is a 'push-pull' electron transfer system from the 5-phenyl moiety to the 2-phenyl ring. The compounds show strong solvent-dependent fluorescence that is well correlated with the empirical solvent polarity parameter E_T(30). These dyes have also shown long emission wavelengths, high extinction coefficients, high quantum yields and large Stokes shifts. Their biological applications will also be discussed.

Although coumarin- and fluorescein-based fluorogenic substrates are widely used in a variety of enzyme assays, they are still ill-adapted for continuous assays in the cases of enzymes that have optimal activity below pH 7.0 because coumarins and fluoresceins generally have the maximum fluorescence above pH 10. Additionally the enzymatic products of coumarin- and fluorescein-based fluorogenic substrates are soluble and readily diffuse away from the site of enzyme activity, and are especially troublesome for in vivo applications. We have now developed a variety of 2-(2'- hydroxyphenyl)-4(3H)-quinazolinone (HPQ)-based enzyme substrates that have overcome these limitations. The water- soluble, colorless, and nonfluorescent substrates generate highly fluorescent and photostable precipitates upon enzymatic action without the addition of the secondary trapping agents. Such fluorogenic precipitating substrates ensure fluorescent histochemical imaging of enzymes with cytologically fine resolution and sharp contrast over cellular autofluorescence and therefore inherently enhance sensitivity and specificity of the targeted analysis.

A metal ion sensitive, fluorescent lipid-bilayer material (5% PSIDA/DSPC) was successfully immobilized in a silica matrix using a tetramethoxysilane (TMOS) sol-gel procedure. The sol- gel immobilization method was quantitative in the entrapment of self-assembled lipid-bilayers and yielded thin films for facile configuration to optical fiber platforms. The silica matrix was compatible with the solvent sensitive lipid bilayers and provided physical stabilization as well as biological protection. Immobilization in the silica sol-gel produced an added benefit of improving the bilayer's metal ion sensitivity by up to two orders of magnitude. This enhanced performance was attributed to a preconcentrator effect from the anionic surface of the silica matrix. Thin gels (193 micron thickness) were coupled to a bifurcated fiber optic bundle to produce a metal ion sensor probe. Response times of 10 - 15 minutes to 0.1 M CuCl₂ were realized with complete regeneration of the sensor using an ethylenediaminetetraacetic acid (EDTA) solution.

Laser desorption mass spectrometry (LDMS) has been developed for DNA sequencing, disease diagnosis, and DNA fingerprinting for forensic applications. With LDMS, the speed of DNA analysis can be much faster than conventional gel electrophoresis. No dye or radioactive tagging to DNA segments for detection is needed. LDMS is emerging as a new alternative technology for DNA analysis.

We report capillary electrophoretic separation of pUC8 and pBr322 plasmid topoisomers in cross-linked polyacrylamide (PAA) gels in 1X TBE buffer. Plasmid topoisomers are supercoiled forms that have exactly the same chain length but differ in their number of superhelical turns. Because the size in base pairs is invariant, topoisomer mobilities reflect conformational details and differ by only small increments. In cross-linked PAA rapid topoisomer separation can be achieved by DC electrophoresis in capillary lengths as short as 3 cm and near-baseline resolution in longer capillaries. We propose that the separation depends upon the regular structure obtained when a gel is prepared intra-capillary. The isothermal environment promotes formation of a cross-linked polymer of low polydispersity. Such PAA is a sieving matrix of high resolving power, but usable over a relatively narrow DNA size range. It is also possible to prepare gels in which a wide base pair range of supercoiled and nicked plasmids as well as linear ds-DNA may be separated, but without topoisomers resolution. In this paper, we discuss the latest results in topoisomer resolution using a range of plasmids employed in molecular biology and gene therapy.

Our current experiments further the development of a laser- based technique capable of sequencing an individual strand of DNA. We report the detection and identification of fluorescently labeled nucleotides enzymatically cleaved from DNA strands suspended in flow. We used fluorescence lifetime, fluorescence intensity, or a correlated measure of the intensity and lifetime to identify each individual tagged base traversing the detection region with high accuracy. DNA strands containing a single tetramethylrhodamine labeled uracil and/or a single Rhodamine 6G labeled cytosine were attached to polystyrene microspheres. An optical trap was used to capture and hold a single DNA-laden microsphere nominally 20 microns upstream of the detection region of an ultra- sensitive flow cytometer. The addition of an exonuclease cleaved bases from the 3' end of the fluorescently labeled strand. The cleaved, labeled nucleotides were carried by the flow downstream and detected and identified one-at-a-time with high efficiency by laser-induced fluorescence.

Dye-labeled dideoxy terminators are the preferred reporter groups for DNA sequencing. This labeling technique can result in uneven peak heights in the electropherograms due to sequence context effects on the biochemical distribution of extension vs termination events. Large variations in peak heights can result in difficulty in identifying very small peaks adjacent to big peaks. Using substrate structure to minimize the sequence context effects on dye-terminator incorporation, we have studied the effects of combinations of base, dye, and dye linkage on terminator incorporation during sequencing. We have identified a propargyl ethoxyamino linker that proved useful in improving peak height evenness and enhancing the terminator activity. New dye-linker-base combinations were optimized for overall peak evenness and relative electrophoretic mobility shifts. The more even peak patterns improve base calling and heterozygote analysis using dye terminator chemistry.

DNA sequencing and several other applications of single- molecule detection (SMD) currently under development utilize spectroscopic measurements for categorization of different types of fluorophores. In the collection and analysis of data from such experiments, the photon signals are sorted into different channels, depending upon their arrival time, emission wavelength, or other distinguishable properties. If the photon statistics are adequate, maximum-likelihood estimation (MLE) techniques can be successfully applied to determine which fluorophore is present. However, data analysis using neural network (NN) methods can offer several advantages. We consider data from a Monte Carlo simulation of SMD in a flow-cell, in which a time-resolved fluorescence decay profile is accumulated for each photon burst. A 2-layer NN, with sigmoid as the activation function, is trained on a set of simulated data using back-propagation and the (delta) - learning rule, and then used for identification of photon bursts in subsequent simulations. The NN is able to consider additional input parameters, such as the amplitudes of the weighted-sliding-sum digital-filter output of the photon bursts and the durations of the bursts. It can yield superior identification of photon bursts, particularly in cases where the fluorophores have disparate fluorescence quantum efficiencies, absorption cross-sections, or photodegradation efficiencies, or where the categorization includes other possibilities, such as background fluctuations, or the simultaneous presence of both fluorophores.

We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nano-reactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also, PCR reactions. These nano-reactors consist of fused silica capillary tubes (length equals 10 - 20 cm; id equals 20 - 50 micrometer) with fluid pumping accomplished using the electro-osmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors were interfaced to the micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X- ray etching (width equals 50 micrometer; depth equals 50 micrometer) and were situated directly on the PMMA-based microchip. This chip also contained an injector, separation channel (length equals 6 cm; width equals 30 micrometers; depth equals 50 micrometers) and a dual fiber optic, near- infrared fluorescence detector. The sequencing nano-reactor used surface immobilized templates attached to the wall via a biotin:streptavidin:biotin linkage produced by PCR using a biotinylated forward primer. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nano-reactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be injected into a capillary tube or a micro-chip device for fractionation. In all cases, the detection of the oligonucleotides was accomplished using ultra-sensitive near- IR fluorescence detection.

A simple apparatus for time-correlated single photon counting (TCSPC) measurements in the near-infrared (near-IR) region for scanning-type applications has been constructed and examined. The apparatus consisted of five major components including a pulsed diode laser source (lasing wavelength equals 780 nm; repetition rate equals 80 MHz; power equals 5 mW; pulse width equals 150 ps), an integrated microscope, a large photoactive area avalanche photodiode (APD), a TCSPC PC-board including the electronics and a windows-based software package for accumulating the fluorescence decay profiles. The instrument response function of this assembly was found to be 460 ps, which is adequate for measuring lifetimes with (tau) _f greater than or equal to 500 ps. Due to the small size of the device, it also allowed implementation into scanning experiments where lifetimes were measured. To demonstrate this capability, a three-well microscope slide containing a near-IR dye was scanned. The decay profile of the near-IR dye, aluminum 2,3-naphthalocyanine, was collected and analyzed to obtain its lifetime, which was found to be 2.73 ns, in close agreement to literature values for this particular dye. In addition, a three dimensional image of aluminum 2,3- naphthalocyanine fluorescence decays was acquired by scanning the microscope head over this three-well glass slide. In the scanning mode, the IRFs as well as the decays of the dyes were found to be very stable. The device demonstrated a concentration detection sensitivity of 2.33 nM, however, the dynamic range was limited due to the APD and its slow time constant (passive quenching). In addition, this microscope head was installed in a LI-COR DNA Sequencer 4000 for collection of scanning images.

Molecular Beacon hairpin shaped fluorescent oligonucleotide probes are powerful tools for quantifying specific nucleic acid sequences. Stratagene is developing a sensitive system, using these probes, for detecting and quantifying initial template copy number of nucleic acid sequences in real time during PCR amplification. The system allows parallel multiple fluorophore detection for many applications including allele discrimination and quantitative gene expression analysis. This instrument, combined with Stratagene's Sentinel Molecular Beacon kits, provides an effective system for molecular biology research. We report here the design and utility of an instrument that combines the capabilities of a microplate fluorescence reader with a PCR thermocycler into a low cost real time detection system. The instrument integrates a multiple fluorophore parallel fiber optic excitation and emission detection system, a precision X-Y translation stage, and a high performance thermoelectric temperature cycler with a computer controlled data collection and analysis system. The system uses standard PCR tubes, tube strips, and 96 well plates as the sample format. The result is a low cost, reliable, and easy to use system with premium performance for nucleic acid quantification in real time. 10

The recent development of cDNA microarray allows ready access to large amount gene expression patterns for many genetic materials. Gene expression of tissue samples can be quantitatively analyzed by hybridizing fluor-tagged mRNA to targets on a cDNA microarray. Ratios of average expression level arising from co-hybridized normal and pathological samples are extracted via image segmentation, thus the gene expression pattern are obtained. The gene expression in a given biological process may provide a fingerprint of the sample development, or response to certain treatment. We propose a K-mean based algorithm in which gene expression levels fluctuate in parallel will be clustered together. The resulting cluster suggests some functional relationships between genes, and some known genes belongs to a unique functional classes shall provide indication for unknown genes in the same clusters.

A novel detection scheme in which DNA hybridization can be detected without target-labeling and washing steps was proposed. In this scheme, the free end of oligonucleotide probes immobilized on a streptavidin-coated cover glass was fluorescently labeled. Changes in the fluorescence anisotropy of the probes upon target hybridization were easily monitored. This scheme could easily be incorporated into a DNA chip or a high-density array. The fluorescence anisotropy was measured by using a fluorescence microscope equipped with polarizing devices and an image intensified CCD camera. The region where probes were hybridized with targets showed an increase in fluorescence anisotropy, which indicates that the interaction between the probe and target resulted in a concomitant change in the rotational relaxation time of fluorescent molecules and the measured fluorescence anisotropy.

A Stanford Research Systems SR844 lock-in amplifier was used to build a sub $10,000 phase-modulation fluorometer capable of measuring nanosecond fluorescence lifetimes. The lock-in directly provided both the DC bias and the AC signal used to modulate the intensity of a blue LED excitation source. A photomultiplier tube measured the emission, and the resulting signal was sent back through a DC block to the lock-in with no external signal processing or heterodyning required. A simple computer program was developed to automate the measuring process and correct for the most common sources of error, namely coherent pickup and stray ambient light. Several standard fluorophores were measured, and the results compare favorably with those from a research grade cross-correlation phase fluorometer up to frequencies of 100 MHz. This system can operate in several configurations, each with benefits and limitations. The system is particularly well suited for fluorescence lifetime based sensing applications, demonstrated by measuring dissolved carbon dioxide online in a bacterial fermentation.

The rational design of small molecules for the selective complexation of analytes has reached a level of sophistication such that there exists a high degree of prediction. An effective strategy for transforming these hosts into sensors involves covalently attaching a fluorophore to the receptor which displays some fluorescence modulation when analyte is bound. Competition methods, such as those used with antibodies, are also amenable to these synthetic receptors, yet there are few examples. In our laboratories, the use of common dyes in competition assays with small molecules has proven very effective. For example, an assay for citrate in beverages and an assay for the secondary messenger IP₃ in cells have been developed. Another approach we have explored focuses on multi-analyte sensor arrays with attempt to mimic the mammalian sense of taste. Our system utilizes polymer resin beads with the desired sensors covalently attached. These functionalized microspheres are then immobilized into micromachined wells on a silicon chip thereby creating our taste buds. Exposure of the resin to analyte causes a change in the transmittance of the bead. This change can be fluorescent or colorimetric. Optical interrogation of the microspheres, by illuminating from one side of the wafer and collecting the signal on the other, results in an image. These data streams are collected using a CCD camera which creates red, green and blue (RGB) patterns that are distinct and reproducible for their environments. Analysis of this data can identify and quantify the analytes present.

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Steady state and time resolved confocal fluorescence microscopy, using a point scanning system, is applied to an investigation of the early stages of photo-induced changes in 3T3-L1 murine fibroblasts using di-sulphonated aluminum phthalocyanine (AlPcS₂) as a photosensitizer. A comparison is made with data obtained using a line scan system and V79-4 Chinese hamster fibroblasts. The steady state data obtained in this work demonstrate that intracellular AlPcS₂ fluorescence intensity increases progressively on photoirradiation. Time-resolved studies indicate that this could result from a progressive decrease in the concentration of the self-quenched membrane-associated form of AlPcS₂ following its conversion into the fluorescent monomeric form.

Presently two methods in concert, a marker method and an electronic monitoring method, have been emphasized for an objective monitoring of drug compliance in ambulatory care. While the marker method proves dose ingestion, the electronic monitoring method provides continuous record of timing of presumptive drug doses. The marker method is however time intensive with associated safety problems, and the electronic monitoring method is easily defeated. We here present preliminary results on modulation sensing, a new method that could be used to non-invasively monitor patient compliance. Measurement is based on observing the amplitude modulation of the emission from both a short lifetime marker fluorophore of interest and a long lifetime reference fluorophore contained in the monitoring device. At some intermediate frequencies, the observed modulation of the combined emission is nearly equivalent to the fractional intensity of the marker fluorophore. This method precludes problems associated with measuring fluorescence intensities in highly scattering media. Using this method we measured the presence of rhodamine 800 (Rh800) in intralipid suspensions and chicken tissue. Rh800 is excited at long wavelengths not absorbed by tissues. Micromolar concentrations of this dye were detected in intralipid and chicken muscle covered with a layer of chicken skin.

Several luminescent complexes of osmium (II) containing polypyridine ligands have been prepared. The syntheses, photophysical and fluorescence polarization properties of [Os(phen)₂(aphen)]²⁺, [Os(tpy)(mcbpy)(py)]²⁺, [Os(ttpy)₂]²⁺, [Os(tpy)(triphos)]²⁺ and [Os(tppz)₂]²⁺ are reported. Where phen is 1,10-phenanthroline, aphen is 5-amino-1,10-phenanthroline, tpy is 2,2':6,2'-terpyridine, mcbpy is 4-methyl-2,2'- bipyridine-4'-carboxylic, py is pyridine, triphos is bis(2-diphenylphosphinoethyl)-phenyl phosphine, ttpy is 4- tolyl,2,2':6,2'-terpyridine, and tppz is 2,3,5,6- tetrakis(2-pyridyl)pyrazine. The complexes absorb light at above 550 nm, emit above 700 nm, and have emission lifetimes longer than 50 ns in water. The emission of all the complexes is polarized, so they can have applications as red light excitable dyes for biophysical studies of macromolecules and for polarization immunoassays. The complexes can also be used for lifetime-based oxygen sensing using low-cost phase fluorometry and a LED light source.

A new highly luminescent 'molecular light switch' has been prepared using osmium (II) metal and nonchromophoric phosphine (dppe) and chromophoric (dppz) ligands to study the dynamics of DNA, where dppe is cis-1,2-bis(diphenylphosphino)-ethylene and dppz is dipyrido[3,2-a:2',3'-c]phenazine. The complex, [Os(dppe)₂(dppz)]²⁺, shows a quantum yield in acetonitrile about twofold larger than analogous ruthenium compound, [Ru(bpy)₂(dppz)]²⁺. The anisotropy of [Os(dppe)₂(dppz)]²⁺ was found to be near 0.2 at -60 degrees Celsius in glycerol. This compound emits at 610 nm on exciting at metal to ligand charge transfer (MLCT) band (390 nm). The lifetimes are 550 and 315 ns in argon and air equilibrated acetonitrile samples respectively. This new class of fluorophore using MLCT complex is useable for measurement of DNA dynamics over a larger time scales, which is not possible with conventional DNA dyes, such as ethidium bromide or acridine derivatives.

Abstract not available.