The absorption of biomolecules on surfaces is a perennial and general problem relevant to fields as different as biomaterials, biosensors, microarrays for proteomics and microfluidics. The 'responsiveness-to-surfaces' character of the proteins augments the difficulty of the general problem of biomolecule adsorption on surfaces. This complexity of protein adsorption generated a large number of contributions over the years, with a rate not showing signs of slowing down. This contribution proposes the use of existent data, as well as data generated in 'programmatic', factorial experiments, to generate scaling relationships regarding protein attachment on polymer surfaces linking protein molecular descriptors, surface descriptors and solution descriptors in a common, quasi-empirical 'engineering' relationships. These relationships can be used for the design of surfaces for proteomics microarrays and microfluidic devices.

Quantifying hybridization and therefore fluorescence signals has become a key-issue in DNA-chip technology. Thus a better understanding of fluorescence near a surface has become a necessity. To study this issue, we modeled the fluorophore after an electromagnetic dipole radiating over the substrate; we then developed a simulation code which enabled us to calculate the observation-angle-dependent-intensity radiated by a population and altitude. In the mean time we developed a polarized-gonio-fluoimeter which permits angular fluorescence patterns and fluorescence polarization measurements. We studied DNA-chips obtained by covalent grafting of labeled oligonucleotides. Simulation curves perfectly matched experimental ones, enabling an accurate determination of fluorophore localization on the substrate. Once achieved a better understanding of the fluorophore emission, we designed and realized a thin-film-coated microscope slide dedicated to the enhancement of DNA-chip fluorescence. This substrate was used in a c-DNA gene analysis. Fluorescence enhancement was clearly observed enabling the detection of Cart1 and P2A which are undetectable when using non-coated microscope slides.

We adapted the nanoparticle-labeling technique from microscopical applications for DNA-chip detection. Nanoparticles can be detected by simple optical means, and exhibit a high stability. So alternative optical readout devices can be applied for the detection of specific DNA- binding on microstructured spots of complementary, surface- immobilized capture DNA. Several devices were tested, and the binding of gold-labeled DNA to complementary and non- complementary sequences was investigated using optical detection of the gold-labeled substrates before and after silver enhancement.

Parallel immunoassay enable simultaneous detection of many analytes forma small volume of sample fluid. The use of exceedingly small quantities of capture antibody immobilized in microspots facilitates ambient analyte assay conditions. Under these assy conditions, the fractional occupancy of capture antibody is independent of sample volume. Analyte concentrations are thus measured with greater sensitivity and potentially greater speed. The small drop size delivered by the Packard BioChip Arrayer, a non-contact piezoelectric robotic dispenser, enables the production of arrays consisting of high reproducible microspots of antibodies. The Packard HyrdoGel coated slide is a porous substrate based on a polymer matrix that provides a 3D hydrophilic environment similar to free solution suitable for biomolecular interactions. This substrate has been used to develop fluorescence-based detection immunoassay in multiplex with high sensitivity and offers advantages of increased probe capacity, functionality and accessability for other biomolecular screening applications. Images are captured with the ScanArray confocal laserscanner, which offers the use to choose from a wide range of fluorescent dyes with high resolution and sensitivity, and analyzed with QuantArray microarray analysis software, which enables researchers to easily and accurately visualize and quantitate gene expression and proteomics data.

Poly-tert-butyl methacrylate-co-methyl methacrylate thin film surfaces were patterned and subjected to surface treatment by UV radiation and NaOH exposure, in order to tailor hydrophilic/hydrophobic conditions. The polymer has potential applications as elements of advanced biosensors and other bio-active devices. The topographies and surface chemistries on the micro- and meso-scales of thin films have been characterized by scanning force microscopy operated in the normal contact imaging mode as well as the lateral force and force versus distance modes.

Interferometers have maximum linear sensitivity to small optical perturbations at the half intensity point defined by quadrature when the signal and reference waves are out of phase by ninety degrees. Under this conditions optical path changes down to a billionth-lambda can be detected. We describe the fabrication of interferometric microstructures on silicon all operating at quadrature, which can have sub- micron dimensions. These dimensions could enable, in principle, the fabrication of over a billion interferometer elements on a disk the size of a CD. In addition, a spinning disk of these elements could have the capacity for mega- samples per second sampling rate. As a step towards that goal, we fabricated a 3-inch silicon wafer with 1024 micro strips of gold, each 20 micro strips of gold, each 20 microns wide and 3 cm long arranged radially. The gold was evaporated to a thickness of 79.1 nm putting them at quadrature for read-out with a 632.8 nm He-Ne laser. Bovine Serium Albumin (BSA), was immobilized on the gold micro strips on half of the wafer which had been treated with Hexadecanethiol. The presence of immobilized BSA modified the lobe structure of the far-field diffraction from the micro strips, we are currently extending these static detection result to high-sampling rates on a spinning disk.

The use of DNA microarrays for analysis of complex biological samples is becoming a mainstream part of biomedical research. Microarrays and gene chips containing a lot of specific polynucleotide sequence of both and novel, can be used to establish the expression of numerous genes in a single experiment. We developed a multicolor fluorescent microbeads for differential display method that combines properties of both differential signal amplification and DNA microarray in signal analysis. The multicolor fluorescent differential display provides a basis upon which a sensitive and accurate gene expression process can be automated and digitally analyzed. The ability to perform such analysis for extremely large nu8mbers of probes on an inexpensive and well-defined high-throughput platform is highly desirable. Microbead-based libraries are emerging as a very attractive alternative smaller probe sites. Most impressive advantages of microbeads are that they inexpensive to produce in a large numbers, they can be conveniently stored in a small volume of fluid, and they can be easily bar coded and screened using a variety of technologies. We developed a dextran-based microbeads containing covalently coupled fluorescent compounds. By combination of different compounds attached to dextran molecules it was possible to increase the number of 'fluorescently barcoded beads'.

Previous attempts have been made to integrate gene amplification (PCR) with electrophoretic separation of PCR products on a chip fabricated in PDMS. PDMS is a widely used elastic polymer for rapid prototyping using soft lithography. In its pre-cure stage, PDMS consists of multiple components and after curing, the polymerization reactions are usually not competed. Consequently, there are low molecule weight compounds present in the PDMS replica. Environmentally dependent surface changes as well as surface contamination for many polymers are well known. All these lead to changes in surface properties. In this study, the surface properties of PDMS were studied. We have detected the presence of volatile oligosiloxanes in the interface between a PDMS replica and a glass substrate. Also, their behavior at room and elevated temperatures, in particular the adhesion strength between the PDMS-glass interface, was analyzed.

Microarray measurements offer the potential to compare the abundances of numerous nucleic acid sequences in parallel. Using linker-adapter PCR products from mapped BAC clones we have made arrays that permit scanning the human genome for single copy gains and losses of DNA sequence, which requires reliable detection of 50 percent changes. The DNA is printed at high concentration on amino-silane or chromium coated surface using a custom-built capillary pin printing system. Spots are printed on 130 micrometers centers or closer to minimize the size of the arrays. Hybridization occurs in a dextran sulfate/formamide buffer at 37 degrees C, using slow rocking to mix the reaction. The entire array is imaged in a single CCD frame using a custom built system that employs mercury arc illumination. Up to four fluorochromes can be imaged from a single array with adequate spectral separation. Typically we use DAPI to stain the DNA in the array spots to facilitate automatic image segmentation during analysis, and fluorescein, Cy3, and Cy5 or their spectral equivalents, for labeling specimen nucleic acids. Array spots are segmented and quantitative fluorescence intensities and intensity ratios are automatically calculated in < 1 minute per approximately 8000 element array using the custom software UCSF SPOT.

New Geiger Mode Avalanche Photodiodes (GM-APD) have been designed and characterized specifically for use in microarray systems. Critical parameters such as excess reverse bias voltage, hold-off time and optimum operating temperature have been experimentally determined for these photon-counting devices. The photon detection probability, dark count rate and afterpulsing probability have been measured under different operating conditions. An active- quench circuit (AQC) is presented for operating these GM- APDs. This circuit is relatively simple, robust and has such benefits as reducing average power dissipation and afterpulsing. Arrays of these GM-APDs have already been designed and together with AQCs open up the possibility of having a solid-state microarray detector that enables parallel analysis on a single chip. Another advantage of these GM-APDs over current technology is their low voltage CMOS compatibility which could allow for the fabrication of an AQC on the same device. Small are detectors have already been employed in the time-resolved detection of fluorescence from labeled proteins. It is envisaged that operating these new GM-APDs with this active-quench circuit will have numerous applications for the detection of fluorescence in microarray systems.

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

The adsorption of biomolecules on surfaces is dependent on biomolecule molecular descriptors, surface descriptors and environment descriptors. Of these descriptors, the biomolecular-related are the most complex and arguably the most important, as being related to both adsorption and possible denaturation on surfaces. The criticality of biomolecule adsorption in the context of microarrays and microfluidics devices will increase with the advancement of proteomics that probes the functionality of proteins, which are much more responsive to surfaces than DNA - the focal biomolecule of genomics. Anticipating this development, the present contribution proposes the establishment of a biomolecular descriptors database, which could be the starting point for a depositary of data and a source of validation for predictive models for adsorption and denaturation.

This paper explores the use of photo patternable polymers for integrated high-speed screening arrays, where enzyme reactions are monitored in nano liter volume reactors using fluorescence of NADH and photodiode detection. Implementing the array of nano liter volume wells using a low-temperature CMOS-compatible process allows wells to be patterned after the photodiode array and electronics fabrication is completed. We demonstrate filling of 400 X 400 micron square, 25 micron deep photoresist-on-silicon wells with liquid samples by electro spray and wetting. We also demonstrate usability of the wells on NADH samples by measuring the fluorescence of 0.1, 0.5 and 1 millimolar NADH solutions using external optics.

The possibility of using microarray technology for mechanistic understanding of drug toxicity has opened up a new research field in Toxicology. In an attempt to build knowledge in the field, we have designed a 1C-gene array composed of 85 known human genes with toxicological interests and 15 control genes. HepG2 cells were treated with ethanol and two anticancer drugs, mitomycin C and doxorubicin. RNA were isolated and labeled by fluorescent dyes, then hybridized to the 1C-gene array. Our results showed that a number of cytochrome P450 genes, such as CYP4F2/3, CYP3A3, CYP24, and CYP51, were consistently responsive to the toxicant treatment. However, different genes response to different toxicants. For example, CYP24 and CYP51 were up regulated by the ethanol treatment but remained unresponsive to the other two drugs. The anticancer drugs, but not ethanol differentially regulated several other genes including CYP3A3, TNFRSF6 and CHES1, implying that the two drugs might function through a similar mechanism, which differs from that of ethanol. The reproducibility of our results suggests that microarray- based expression analysis may offer a rapid and efficient means of assessing drug toxicity.

G protein-coupled receptors (GPCRs) are the largest family of cell surface proteins involved in transmitting extracellular signals to the interior of the cell. These membrane-spanning proteins constitute one of the most important families of drug targets. Despite their importance, the power and utility of microarray technology has not been extended to GPCRs or other membrane proteins because of issues due to immobilization - these proteins typically need to be embedded in membrane environment to maintain their native conformations. This paper describes the fabrication of GPCR microarrays by conventional robotic pin-printing and demonstrates straightforward assays for screening of ligands on these arrays. GPCRs, obtained as membrane preparations form cell lines over-expressing particular GPCRs, were arrayed using a quill-pin printer. The arrays were incubated with solutions of labeled cognate ligands and unlabeled compounds, and imaged using a fluorescence scanner. The assays conducted were designed to test: (i) the specificity of ligand binding among different families of GPCRs; (ii) the selectivity of ligand binding and inhibition among different members of a GPCR family; (iii) the affinity of ligand binding. The results showed highly selective binding of ligands to arrays of receptors, with affinities similar to those reported in the literature and obtained suing other techniques. This demonstration of membrane-protein arrays and associated assays overcomes a fundamental limitation in protein microchip technology - the lack of practical microarray based methods for membrane proteins.

Near-IR Spectroscopy (NIRS) is used extensively in the health services industries: medical research, pharmaceutical production, and bioprocessing. NIRS Is rugged, simple to operate, flexible, and relatively inexpensive. It may be used to monitor the progress biochemical reactions. It is used to control mixing, blending, drying, and coating in pharmaceutical production and is used for imaging and chemical determinations in living patients.

Spectroscopic studies have been done on crystallization and other reactions that undergo multiple phase changes. As the reaction opacity changes the spectral information is processed to determine which mode of measurement is optimal: reflectance or interactance immersion. A near IR spectrophotometer equipped with a unique probe is used which allows for optimal process monitoring with a single probe, rather than using multiple process interfaces to accommodate the change in the reaction medium. A with any near-IR method, models are created to allow for reproducible predictions of the phase changes that occur, and can also be used to quantitate the extent of reaction, the system composition, and other parameters.

The sensitivity and spatial resolution of hyperspectral imaging instruments are tested in this paper using pharmaceutical applications. The first experiment tested the hypothesis that a near-IR tunable diode-based remote sensing system is capable of monitoring degradation of hard gelatin capsules at a relatively long distance. Spectra from the capsules were used to differentiate among capsules exposed to an atmosphere containing imaging spectrometry of tablets permits the identification and composition of multiple individual tables to be determined simultaneously. A near-IR camera was used to collect thousands of spectra simultaneously from a field of blister-packaged tablets. The number of tablets that a typical near-IR camera can currently analyze simultaneously form a field of blister- packaged tablets. The number of tablets that a typical near- IR camera can currently analyze simultaneously was estimated to be approximately 1300. The bootstrap error-adjusted single-sample technique chemometric-imaging algorithm was used to draw probability-density contour plots that revealed tablet composition. The single-capsule analysis provides an indication of how far apart the sample and instrumentation can be and still maintain adequate S/N, while the multiple- sample imaging experiment gives an indication of how many samples can be analyzed simultaneously while maintaining an adequate S/N and pixel coverage on each sample.

Fluorescence detection is one ofthe most popular detection methods used in bioanalytical analysis. Fluorescence detection methods offer the advantages ofhigh sensitivity combined with selectivity. Generally, the chromophore is attached to the species of interest and acts as a reporter molecule. Most fluorescence detection schemes involve the use ofvisible fluorophores, fluorescein being one ofthe most common. A problem often encountered with the used of fluorescence detection in the visible region of the spectrum is sample autofluorescence. Many compounds have intrinsic fluorescence in this region. Background fluorescence is present throughout the visible and UV region ofthe spectmm (fig 1). Consequently, background noise can be very high and, as a result, the overall sensitivity of the measurement is decreased. Very few molecules exhibit intrinsic fluorescence in the near infrared region of the spectrum (650-1 100 nm). As a result, the background noise associated with fluorescence detection in the visible region is nearly eliminated. Consequently, the near infrared region offers the potential for significant improvements in sensitivity, especially in situations where matrix autofluorescence is a concern. Near infrared fluorescence detection methods are therefor well suited to bioanalytical applications. Furthermore, light scatter, another source of interference, is decreased in the near infrared region due to its ii)dependence. Detection at 820 nm offers a six-fold reduction in light scatter over detection at 500 nm. Instrumentation associated with the use of near infrared dyes also offers some advantages over traditional instrumentation. Laser induced fluorescence is one ofthe most commonly used fluorescence detection methods, due to the high intensity associated with laser light. Lasers that operate in the visible region ofthe spectrum are often bulky, expensive, and have limited operational lifetimes. The advent of solid state diode lasers do not have any of the aforementioned disadvantages. They are mgged, inexpensive, compact, and have long operational lifetimes. The typical signal transducer for fluorescence measurements is the photomultiplier tube (PMT). However, PMT's make poor choices for signal transducers in the near infrared region due to their low quantum yields at these wavelengths. Consequently, Avalanche photodiodes (APD) are used. APD's offer high quantum efficiencies in the near infrared region. Additionally, they are rugged, cheap, and have long operational lifetimes.

A developmental optical tomography has been designed for imaging small animals in vivo using near IR fluorophores. The system employs epi-illumination via a 450 W Xe arc lamp, filtered and collimated to illuminate a 10 cm square movable stage. Emission light is filtered then collected by a high- resolution, high quantum efficiency, cooled CCD camera. Stage movement and image acquisition are under the control of a personal computer running system integration and automation software. During an experiment, the anesthetized animal is secured to the stage and up to 200 projections can be acquired over 180 degrees rotation. Angular sampling of the light distribution at a point on the surface is used to determine relative contributions form ballistic and diffuse photons. We have employed the system to investigate a number of applications of in-vivo fluorescent imaging. In dynamic studies, hepatic function has been visualized in nude mice following intravenous injection of indocyanine green (ICG) and cerebrospinal fluid flow as been measured by injection of ICG-lipoprotein conjugate in the subarachnoid space of the lumbar spine followed by dynamic imaging of the brain. Further applications in physiological imaging, cancer detection, and molecular imaging are under investigation in our laboratory.

The use of fluorescently labeled probes for membrane-based analysis of proteins and nucleic acids have been unsuccessful due to high background fluorescence of the membranes. We have developed a system for the analysis of proteins and nucleic acids using near infrared (IR) fluorescent dyes and a new scanning instrument, the Odyssey Infrared Imager. Nucleic acid probes can be directly labeled with carbodiimidederivatized IR dyes in a 10 minute labeling and clean up procedure. In a Northern blot format, mRNA from genes such as mouse 3-actin, mouse GAPDH, and cyclophilin mRNA have been detected in as little as 0.1 tg of total RNA. In immunoblotting assays, JR detection of proteins was as sensitive as chemiluminescence and routinely enabled detection of 1-10 pg of protein. Signal transduction events in the MAP kinase pathway were analyzed using two-color immunoblotting. Phosphorylated and non-phosphorylated proteins were detected and quantitated simultaneously without the need for stripping and reprobing. A model system for assessing protein-nucleic acid interactions by EMSA was developed based on the binding of T7 RNA polymerase to its promoter. Varying amounts of protein were combined with a PCR-generated IRD800labeled DNA fragment. Protein binding could be monitored quantitatively following scanning of the gel on the Odyssey Infrared Imager. The Odyssey Infrared Imaging system permits two-color analysis of proteins and nucleic acids with very high signal to noise ratios.

Oxygen dependent quenching of phosphorescence is a powerful method for measuring oxygen. Phosphors are now available that absorb and emit in the near IR region of the spectrum, are nontoxic, and remain in the blood, allowing rapid measure of oxygen through out selected tissue volumes. In vivo measurements are non-invasive except for the need to inject phosphor into the blood, and phosphorescence lifetimes can be measured without interference by tissue pigments that absorb or fluorescence at the measurement wavelengths. Phosphorescence quenching is uniquely useful for: (1) imaging oxygen in optically clear media or in the surface layer of the tissue, such as in the retina of the eye; (2) determining the distribution of oxygen in media, such as tissue, which have heterogeneous distributions by deconvoluting phosphorescence decay dat. These can be used to calculate the corresponding oxygen histograms. Measurement in 2D grids can b used to construct contour maps of the fraction of the sampled tissue volume with any selected range of oxygen pressures. These maps accurately show the location and size of any regions of hypoxia within the sampled tissue.

Phosphorescence quenching is an optical method for measuring tissue oxygenation. The technique is based on the quenching of phosphorescence originated from the injected dye by molecule oxygen dissolved in the medium. The phosphor is the only 'invasive' component of the measurement procedure, and thus it is important to have precise control over the bio- distribution of the phosphor, i.e. to confine it to a single compartment within the sample. For tissue applications the phosphor must also be an effective light absorber in the near IR and to exhibit oxygen quenching constant of 200-400 Torr^-1 sec^-1, to permit reliable quantification of oxygen in arterioles as well as in veins. Overall, it is desirable to have synthetic, inert, hydrophilic, phosphors with quenching characteristics that are not affected by molecules other than oxygen. We discuss a new generation of phosphors based on dendrimer- tetrabenzoporphyrins, designed to satisfy the above criteria. In these phosphors, the core metallotetrabenzoporphyrins prove the required physical characteristics, while their immediate surrounding environments consist of covalently attached dendritic branches. The dendritic cages around porphyrins control their quenching properties and protect porphyrins from interactions with other substances in the blood.

While fluorescence continues to be an important tool in genomics, new challenges are being encountered due to increased efforts toward miniaturization reducing detection volumes and the need for screening multiple targets simultaneously. We have initiated work on developing time- resolved near-IR fluorescence as an additional tool for the multiplexed analyses of DNA, either for sequencing or mutation detection. We have fabricated simple and compact time-resolved fluorescence microscopes for reading fluorescence from electrophoresis or DNA microarrays. These microscopes consist of solid-state diode lasers and diode detectors and due to their compact size, the optical components and laser head can be mounted on high-speed micro-translational stages to read fluorescence from either multi-channel capillary electrophoresis instruments or micro fabricated DNA sorting devices. The detector is configured in a time-correlated single photon counting format to allow acquisition of fluorescence lifetimes on-the-fly during data acquisition in the limit of low counting statistics. In multiplexed analyses, lifetime discrimination serves as a method for dye-reporter identification using only a single readout channel. Also, coupled to multi-color systems, lifetime identification can significantly increase the number of probes monitored in a single instrument. In this work, near-IR fluorescence, including dye-labels and hardware, will be discussed as well as the implementation of near-IR fluorescence in DNA sequencing using slab gel electrophoresis and DNA microarrays.

We have developed a novel optical coding technology for massively parallel and high-throughput analysis of biological molecules. Its unprecedented multiplexing capability is based on the unique optical properties of semiconductor quantum dots (QDs) and the ability to incorporate multicolor QQs into small polymer beads at precisely controlled ratios. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic studies indicate that the QD tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99 percent under favorable conditions. DNA hybridization results demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnosis.

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Survival prediction and optimal treatment choice for cancer patients are dependent on correct disease classification. This classification can be improved significantly when high- throughput data such as microarray expression analysis is employed. These data sets usually suffer from the dimensionality problem: many features and few patients. Consequently, care must be taken when feature selection is performed and classifiers for disease classification are designed. In this paper we investigate several issues associated with this problem, including 1) data representation; 2) the type of classifier employed and 3) classifier construction, with specific emphasis on feature selection approaches. More specifically, 'filter' and 'wrapper' approaches for feature selection are studied. The different representations, selection criteria, classifiers and feature selection approaches are evaluated with regard to the effect on true classification performance. As test cases we employ a Comparative Genomic Hybridization breast cancer data sets and two publicly available gene expression data sets.

In this paper we present the innovative use of an inexpensive Multi-Channel Capillary-based Electrophoresis (MCCE) system in combination with disposable separation cartridge for routine analysis of DNA fragments. The proposed multi-channel system s base don a novel multiplexed fluorescence detection technology, which provides a rapid and unique solution for DNA analysis. Presently the Capillary Electrophoresis (CE) based technology used for DNA analysis, rely on gas discharge UV-visible lamps or lasers as light sources that are bulky, expensive, and difficult to couple one's light output into optical fibers, for miniaturization of the optical detection system. The light sources hinder the development of small sized, high- throughput and cost-effective genomics instrument. Whereas, the proposed instrument with solid-state light sources and non-moving detection micro-optics, and re-usable cartridge containing multiple separation channels, provide a cost- effective solution for a robust CE instrument. Furthermore, the simplified operation of the MCCE instrument will drastically reduce the cost of DNA analysis, and possibly will be the instrument of choice for forensic DNA and molecular diagnostics applications in the near future.

A probe DNA monolayer modified with Au nano-particles was prepared on an Au thin film and used for DNA hybridization detection by surface plasmon resonance (SPR) spectroscopy. This Au particle modified probe DNA monolayer resulted in about a 3.5 times larger SPR angle shift according to the hybridization reaction, compared to that found in a non-modified probe DNA monolayer. By comparison of the SPR image of the particle modified and nonmodified probe DNA monolayer, this modification method was shown to be useful in improving the SPR signal for the detection ofthe unlabelled DNA molecules.

Numerous commercial instruments exist for assaying fluorescence signals from an array of samples during thermal cycling and thermal denaturation profiling; however these instruments tend to be complex, expensive, and/or require custom reaction vessels. We have developed a system that incorporates a novel fluorescence excitation scheme in which an independently addressable array of light sources is imaged into the wells of a standard 96-well plate in a sequential fashion and the resultant fluorescent excitation is read by a photomultiplier tube (PMT). By individually illuminating each sample, we have eliminated the need for sophisticated detection and image analysis techniques used to discriminate signals form different samples. Additionally, independent control of each light source allows the system to automatically balance the light incident on each vessel to provide a flat response across the array, thus avoiding any post-measurement normalization that would reduce dynamic range. The resulting instrument has high sensitivity due to the PMT, but low sample crosstalk due to the sample illumination control granted by the LED array. Application data is shown including single copy DNA amplification and detection as well as product thermal denaturation analysis.

Miniaturization of sensitive detection system is a key factor for integrated chemical systems. Thermal lens microscope can be applied to the ultra-sensitive detection of non-fluorescent molecules in many applications. By using a very small SELFOC micro lens as an objective, palmtop- sized thermal lens microscope (PTLM) has been constructed. A calibration curve for an aqueous solution in a range of 1.0 by 10^-7-5.0 by 10^-6 mol/L has been measured in this newly designed PTLM.

The need for higher throughput, higher dynamic range detectors for protein crystallography is stimulating work on new detector concepts. Current detectors such as photo- cathode converters optically coupled to Charge Coupled Devices (CCDs) use a two step detection process. The incoming x-ray is converted in the photo-cathode in low energy photons. These longer wavelength photons can be efficiency detected in the CCD where they create an electrical charge. The overall process is quite inefficient as few photons are generated in the primary converter and fewer are collected in the CCD. Due to the scarcity of photons per x-ray an event driven processing of the information is impossible. New approaches favors the direct detection of the incoming photon using either semiconductor detectors, the most commonly used being silicon, or gas detectors with internal amplification. The incoming photons directly create change in the detector at levels sufficient to be processed on a per event basis. All these approaches share common features: event level processing, large dynamic range, and fast readout leading to high throughput. While the primary application is in photon counting these detectors are also capable of providing multi-parameter data such as the measure of the x-ray energy and its time of occurrence. Several groups are working on the development of these detectors using different approaches to the capture of the information and its readout. The capabilities and limitations of these implementations are reviewed.

A complete, 3D theory of Compton scattering is described, which fully takes into account the effects of the electron beam emittance and energy spread upon the scattered x-ray spectral brightness. In the linear regime, and in the absence of radiative corrections; it is found that each vacuum eigenmode gives rise to a single Doppler-shifted classical dipole excitation. This formalism is then applied to Compton scattering in a 3D laser focus, and yields a complete description of the influence of the electron beam phase space topology on the x-ray spectral brightness; analytical expressions including the effects of emittance and energy spread are also obtained in the 1D limit. Within this framework, the x-ray brightness generated by a 25 MeV electron beam is modeled, fully taking into account the beam emittance and energy spread, as well as the 3D nature of the laser focus; its application to x-ray protein crystallography is outlined.

Circulating p53 autoantibodies are found to be a universal and highly specific tumor marker for malignant diseases. Hence, sereological screening for p53 autoantibodies at low concentration levels has become increasingly relevant for early-stage and follow-up of tumor diagnostics. We developed a new method for the highly sensitive detection of p53 antibodies in a homogeneous fluorescence assay format. Short, linear peptide derived form antibody recognition sequences so human p53 were labeled with an oxazine dye. Hydrophobic interactions constrain a conformation, where the dye interacts selectively with a tryptophan residue in the peptide sequence. Subsequently, the fluorescence of the dye is quenched efficiently due to electron transfer from the indole derivative to the dye in the excited state. Specific antibody recognition induces a conformational change in the peptide structure, repealing the dye-tryptophan interaction. Consequently, a fluorescence increase upon antibody binding signals the binding event. The long-wavelength absorption and emission characteristics of the probe and the use of a red pulsed diode laser as excitation source in a confocal fluorescence microscopic set-up allows ultra sensitive antibody detection at the single-molecule level. The effectiveness of the probes are highlighted by the detection of individual p53 autoantibodies directly in serum dilutions of cancer patients.

As biological analysis systems scale to smaller dimensions, the realization of small and portable biosensors becomes increasingly important. The innovation of integrated fluorescence sensors is now possible due to the development of optoelectronics over the past decade. We present the monolithic integration of vertical cavity surface emitting lasers, PIN photo-detectors and optical emission filters to be used as a fluorescence sensor. The integration will drastically reduce cost and size of fluorescence detection systems. Also, parallel sensing architectures of more than one hundred channels will be possible. The sensor will be utilized for near-IR fluorescence detection. This spectral range is compatible with standard AlGaAs optoelectronic technology and will also reduce background fluorescence from complex bio-fluids such as blood. PIN heterostructure photodetectors have been fabricated and tested. Photodetector experiments show extremely low dark current of less than 500fA/mm, quantum efficiency greater than 85 percent and linear detector response. Optical simulations predict a detection sensitivity lower than 10000 fluorescent molecules in a detection area of 10⁴ micrometers ².

The analysis of DNA fragments by ion-pair reversed-phase high-performance liquid chromatography on an alkylated, nonporous poly(styrene-divinylbenzene) matrix (DNA Cartridge) using the WAVE Nucleic Acid Fragment Analysis System is a powerful and versatile tool for DNA analysis. Resolution of DNA fragments is based on two principles, size-dependent retention of double-stranded (ds) DNA and differential retention of ds vs. single-stranded (ss) DNA. Temperature Modulated Heteroduplex Analysis utilizes both principles of separation to detect single nucleotide polymorphisms (SNP) and short insertions/deletions. At a given temperature the difference in the melting between homo- and heteroduplexes is revealed by differences in retention times. The temperature at which differential melting occurs is sequence dependent and is predicated accurately using either WAVEMAKER or WAVE Navigator software, which use a modified Fixman-Friere algorithm. Detection of known and unknown sequence variations can be performed on DNA fragments of up to 1,000 base pairs with high sensitivity and specificity. The use of fluorescent labels is compatible with the technology and increases sensitivity. Retention times are increased and resolution is not affected. Fluorescent labeling significantly increases sensitivity.

We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.

Pyrosequencing is a DNA sequencing technique that takes advantage of the cooperativity of four enzymes in a single- tube to determine the nucleotide composition of a DNA fragment in real-time. In this manuscript we describe the methodology and the use of this technology for analysis of single nucleotide polymorphisms, although, this technique has also been used for sequence determination of difficult secondary structures, mutation detection, EST sequencing, virus and bacteria typing, and re-sequencing of disease genes. Recent break-through has enabled long read data up to 200 nucleotides to be obtained in a single run. Automated microtiter plate based Pyrosequencing systems have been developed allowing DNA analyses of between 5000 to 50,000 samples per day. We are now miniaturizing this technique to reduce the cost for sequencing by at least two order of magnitudes. The array format proofs the feasibility of this system for DNA sequencing.

Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described. These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 order of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, final load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new T_m prediction software to design robust 5'-MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the T_m of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo(3,4- d)pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation between individuals of a species and are therefore thought to be responsible for a large part of individual phenotypic variation. It has been estimated that a SNP may occur every 100-300 bases in the human genome. Research on human SNPs is expected to facilitate genetic mapping studies that may lead to a better understanding of the genetic basis for complex diseases and individual variation in drug metabolism. We have developed a novel assay for the identification of known SNPs using primer extension with the novel AcycloTerminators and a new thermostable polymerase, AcycloPol, in a homogeneous fluorescence polarization (FP) format. All assay steps can be performed in the same well of either a 384- or 96-well PCR-compatible microplate. FP provides several advantages, including simplicity and low reagent cost. The homogeneous assay format eliminates any need for separation or washing steps and is amenable to automation.

We are investigating optoelectronic properties of integrated structures comprising semiconductor light-emitting materials for optical probes of microscopic biological systems. Compound semiconductors are nearly ideal light emitters for probing cells and other microorganisms because of their spectral match to the transparency wavelengths of biomolecules. Unfortunately, the chemical composition of these materials is incompatible with the biochemistry of cells and related biofluids. To overcome these limitations, we are investigating functionalized semiconductor surfaces and structures to simultaneously enhance light emission and flow of biological fluids in semiconductor micro cavities. We have identified several important materials problems associated with the semiconductor/biosystem interface. One is the biofluid degradation of electroluminescence by ionic diffusion into compound semiconductors. Ions that diffuse into the active region of a semiconductor light emitter can create point defects that degrade the quantum efficiency of the radiative recombination process. This paper discusses ways of mitigating these problems using materials design and surface chemistry.

Yeast-Saccharomyces cerevisiae - it widely used as a model system for other higher eukaryotes, including man. One of the basic fermentation processes in yeast is the glycolytic pathway, which is the conversion of glucose to ethanol and carbon dioxide. This pathway consists of 12 enzyme-catalyzed reactions. With the approach of microarray technology we want to explore the metabolic regulation of this pathway in yeast. This paper will focus on the design of a conventional microscope based microarray reader, which is used to monitor these enzymatic reactions in microarrays. These microarrays are fabricated in silicon and have sizes of 300 by 300 micrometers ². The depth varies from 20 to 50 micrometers . Enzyme activity levels can be derived by monitoring the production or consumption rate of NAD(P)H, which is excited at 360nm and emits around 450nm. This fluorophore is involved in all 12 reactions of the pathway. The microarray reader is equipped with a back-illuminated CCD camera in order to obtain a high quantum efficiency for the lower wavelengths. The dynamic range of our microarray reader varies form 5(mu) Molar to 1mMolar NAD(P)H. With this microarray reader enzyme activity levels down to 0.01 unit per milliliter can be monitored. The acquisition time per well is 0.1s. The total scan cycle time for a 5 X 5 microarray is less than half a minute. The number of cycles for a proper estimation of the enzyme activity is inversely proportional to the enzyme activity: long measurement times are needed to determine low enzyme activity levels.

We are developing a novel readout for secretion of hormones and neurotransmitter on micro/nanofabricated chips. Traditional biochemical assays of signaling molecules secreted from cells are slow, cumbersome and have at best, a temporal resolution of several seconds. On the other hand, electrochemical measurement of hormone or transmitter secretion can obtain millisecond temporal resolution if the diffusion distance between the release site on the cell and the working electrode is within 1 micron. Carbon fiber microelectrodes can have millisecond time resolution, but can only measure release form a small fraction of the cell surface. We have fabricated arrays of Au electrodes in wells micromachined on the surface of silicon microchips. Each well/microelectrode roughly conforms to the shape of a single cell in order to capture release forma large fraction of the surface area of each cell with minimal diffusional delays. This paper will present details of the microfabrication process flow as well a initial results demonstrating millisecond-resolution measurement of catecholamine secretion form adrenal chromaffin cells. Our goal for this project is to develop enabling technology for massively parallel systems on a chip such as cell-based biosensors to detect neurotoxins and high-throughput assays of drugs that affect neurotransmitter release.

Monodisperse, spherical, polyethylene glycol (PEG)-coated silica nanoparticles have been prepared in the size range of 50-350 nm, and their size distribution were characterized by SEM and multi-angle static light scattering experiments. The chemical binding of PEG to the silica nanoparticles was confirmed by IR spectroscopy. The biocompatibility of these PEGylated nanoparticles was also studied by non-specific protein binding tests and in-vivo toxicology studies in live animals. These silica nanoparticles, as a matrix for encapsulation of certain reagents, have been used for the fabrication of intracellular sensors and have potential for applications to in vivo diagnosis, analysis and measurements, due to their small physical size and their biocompatibility, both stemming from the specialized PEG coating.

In this paper the photothermal engineering issues of novel shape memory polymer (SMP) microactuators for treating stroke are presented. The engineering issues for using lasers to heat and subsequently actuate these SMP devices are presented in order to provide design criteria and guidelines for intravascular, laser activated SMP devices. The optical properties of SMP, methods for coupling laser light into SMP, heating distributions in the SMP devices and the impact of operating the thermally activated material in a blood vessel are presented. A total of three devices will be presented: two interventional ischemic stroke devices and one device for releasing embolic coils. The optical properties of SMP, methods for coupling laser light into SMP, heating distributions in the SMP devices and the impact of operating the thermally activated material in a blood vessel are presented. Actuating the devices requires device temperatures in the range of 65 degrees C - 85 degrees C. Attaining these temperatures under flow conditions requires critical engineering of the SMP optical properties, optical coupling into the SMP, and device geometries. Laser- activated SMP devices are a unique combination of laser- tissue and biomaterial technologies. Successful deployment of the microactuator requires well-engineered coupling of the light form the diffusing fiber through the blood into the SMP.

A major challenge in many biosensing applications is the real-time detection of a multitude of analytes from a small sample volume. Achieving these goals would perhaps eliminate the need for an intermediate molecular amplification step. Our approach to this challenge involves the investigation of high sensitivity and scalable integrated optical transduction and scalable microfluidic sample delivery. The microfluidic architecture has small cross-section and allows the sample to visit each sensing zone, where a biospecific monolayer performs molecular recognition. Signal transduction occurs via a resonant optical microcavity, which has the dramatically increased signal to noise ratio in fluorescence detection necessary to detect small molecular numbers. Important performance issues in this architecture are sample flow rates, sensing zone size, and the use of passive mixing structures. In addition, microfabrication issues such as optical and microfluidic design, materials, and monolayer patterning are discussed.

Components of a microspectrometer for operation in the IR range has been designed, fabricated, and characterized. An adjustable Fabry-Perot interferometer is used to select the resonant frequency of the system through electrostatic actuation, allowing tuning for certain optical frequencies to pass. Silicon microfabrication techniques are employed for the fabrication of the device. The intended use of the device is for spectroscopic study of liquids in biomedical and environmental applications; therefore, a sample containment chamber has been integrated into the device. The device was designed using finite element modeling to determine the stress distribution on the silicon nitride membrane due to deflection and the voltage required for the suitable displacement of the membrane to which one mirror is attached. The devices have been fabricated using a combination of processing steps to sputter gold mirrors on nitride membranes, to deposit electrodes and spacers using evaporation and photosensitive polyimide, to etch channels and sacrificial layers, and to bond chips to obtain a resonant cavity. Optical characterization was performed with an FTIR spectrometer. Initial results presented here support the feasibility of the approach in developing standalone microspectrometers for analysis of aqueous samples including biological fluids.

Laminar Fluid Diffusion Interfaces are generated when tow or more streams flow in parallel in a microfluidic structure. This technology can be used for diffusion-based separation and detection applications, for example: DNA desalting, the extraction of small proteins from whole-blood samples, and the detection of various constituents in while blood. Additional applications are the establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients, enabling the uniform and controlled exposure of cells to lysing agents, allowing the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure. We have developed integrated systems using machine-controlled disposable cartridges and passive self-contained disposable cards including particle separators, flow cytometers, valves, detection channels, mixers, and diluters that are used in a hematology analyzer, stand-alone blood plasma separators, and a variety of chemical and biological assays. Microfluidic arrays compatible with common well-plate formats have been designed for high-throughout toxicology screening applications. All these devices were manufactured using Micronics' unique rapid-prototyping process yielding low-cost plastic disposable microfluidic chips.

The micromoulding of polymers has provided an ideal fabrication route for the construction of single-use integrated devices for electrokinetic microseparations. Our designs have incorporated injector geometry that allows variable injection volumes in microchannels controlled by a combination of programmed hydrodynamics and electrophoresis. The utility of the injection scheme is demonstrated for isotachophoresis separations. Further developments have lead to the incorporation of parallel opposed conductivity detection electrodes in two ways. Firstly, by the injection of conducting polymer into a pre-molded channel system and secondly through the molding of the polymer microchannels around electrodes pre-molded in conducting polymer. This has provided a potential rapid manufacturing route for low cost polymer separation devices. As well as integrated conductivity detection electrodes, simple optical elements have been incorporated into injection molded ITP devices to permit detection of the separated bands by optical means. A simple spherical lens was incorporated into the top section of the device which included the sample and buffer reservoirs. The lens was positioned directly above the conductivity detection electrodes, to permit simultaneous electrochemical and optical detection. The lens was used to perform visible absorption spectroscopy using an Ocean Optics spectrography and tungsten-halogen fiber-optic white light source.

We present a new label free optical technique to study biomolecular interactions in real time on a surface. This method monitors changes in the absorbance spectrum of a monolayer of gold on glass as a function of biomolecular binding. Gold nanoparticles with a diameter of 13 nm were chemisorbed onto an amine-terminated glass surface. The absorbance spectrum of the monolayer exhibited both a red shift as well as an increase in the absorbance at peak wavelength as a function of bulk solution refractive index. The increase in absorbance at peak wavelength as a function of bulk solution refractive index. The increase in absorbance at 550 nm was employed to study the kinetics of fibrinogen adsorption on the immobilized monolayer. The result obtained with the absorbance sensor were compared with those obtained using conventional SPR. This sensor is attractive because of its simplicity: gold nanoparticles are easily prepared with high reproducibility, they can be easily immobilized on glass, and their absorbance spectrum can be easily measured using widely available UV-vis spectrophotometers. Furthermore, this technique should be easily amenable to the design of chips in an array format for application in hgih-throughput immunoassays and proteomics.

Up converting chelates are a new type of optical reporter with multiple advantages of biomedical diagnostics. We describe these materials and report spectroscopic properties relevant to their use.

Excitation-contraction (e-c) coupling in atrial myocytes was studied with fast confocal microscopy and simultaneous Ca²⁺-current measurements. Cat atrial myocytes lack transverse tubules and contain junctional (j-SR) and non- junctional SR (nj-SR) which both ave ryanodine receptor Ca²⁺ release channels. I_Ca triggered Ca²⁺ release from discrete peripheral j-SR release sites, which then fused into a peripheral 'ring' of elevated (Ca²⁺)_i, followed by propagation to the center and contraction. J-SR Ca²⁺ release could be terminated instantaneously by interrupting I_Ca, whereas nj-SR Ca²⁺ release continued, once initiated, even after I_Ca and j-SR Ca²⁺ release was terminated. Resting myocytes exhibited spontaneous Ca²⁺ release events including Ca²⁺ release sites. In summary, during e-c coupling in atrial cells the elevation of (Ca²⁺)_i begins with j-SR Ca²⁺ release under the tight control of I_Ca. The peripheral increase of (Ca²⁺)_i subsequently activates adjacent nj-SR resulting in centripetal propagation of activation via CICR.

Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon binding to DNA or RNA. We show that two-photon fluorescence lifetime imaging of SYTO13 allows the differential and simultaneous imaging of DNA and RNA in living cells and allows sequential and repetitive assessment. Two-photon main of SYTO13 exhibits a fluorescence lifetime of 3.4 +/- 0.2 ns when associated with nuclear DNA, while bound to RNA its lifetime is 4.1 +/- 0.1 ns. After the induction of apoptosis, clusters of SYTO13 with a fluorescence lifetime of 3.4 +/- 0.2 ns become apparent int eh cytoplasm, identified as mitochondrial DNA on the basis of localization experiments. Upon progression of apoptosis the lifetime of SYTO13 attached to DNA shortens significantly, while no changes are detected in the lifetime of SYTO13 attached to DNA and RNA we started an in vitro study. Here we report the first results of this study. The binding fraction of SYTO13 to DNA increases with increasing amount of DNA (RNA), reaching a plateau for DNA (RNA) concentrations above 0.5 mg/ml. From the spectral measurements we conclude that the natural fluorescence lifetime (tau) ₀ of SYTO13 attached to DNA is 3.7 +/- 0.2 ns, while that of SYTO13 attached to RNA is 4.4 +/- 0.2 ns.

Fluorescence spectroscopy is a widely used research tool in biochemistry and has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing and genomics. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by a nearby metallic surface, the most important effect being an increase in the radiative decay rate. We describe the usual effects expected form increase in the radiative rates with reference to the biomedical applications of immunoassay and DNA hybridization. We also present experiments which show that metallic particles can increase the quantum yield of low quantum yield fluorophores, increase fluorophore photostability and increase the distance for resonance energy transfer. And finally we show that proximity to silver particles can increase the intensity of the intrinsic fluorescence from DNA.

In this study we show that steady-state fluorescence anisotorpy within PEBBLEs can be used for the optochemical sensing of analytes such as Zn²⁺, O₂, and Ca²⁺. Steady-state fluorescence anisotropy is a non- time resolved method that measures a combination of rotational and fluorescence lifetimes. This eliminates the need for reference dyes and ratiometic techniques to obtain quantitative results, even when using intensity-based sensor dyes. An advantage to working with PEBBLE nanosensors is that the encapsulated dye is localized in a constant rotational environment. This is in contrast to the use of free dyes, which can be affected by interferents such as protein binding.

Excited state absorption (ESA) spectroscopy test electronic transitions from the first excited states to higher ones. These transitions are often much more sensitive to the environment of the specimen under investigation than ground state absorption spectra as well as fluorescence spectra. Therefore ESA on intermolecular interactions becomes a candidate for efficient biochemical sensors. In addition, many applications where fluorescence probes are used today may be improved in future regarding sensitivity and velocity by the use of ESA, because absorptive measurements principally allow much faster detection in comparison to fluorescence. Two examples are given: on one hand photoinduced intramolecular electron transfer reactions in special pyrenylbenzene compounds were proven to be strongly dependent on environmental conditions are polarity, for example. Moreover, intermolecular electron transfer to the neighborhood was identified by this technique. On the other hand the denaturation of myoglobin is observed by ESA spectroscopy on fluorescein probes covalently bound to be protein. During unfolding the protein the local environment of the probes changes, which si reflected effectively in the spectra. In both examples the ESA method is compared to traditional fluorescence techniques. Besides the confirmation of the traditionally obtained results, valuable new information is yielded.

A novel Charge Coupled Device (CCD) has been commercially produced by Marconi Applied Technology, UK under the trade name of L3Vision, incorporating a solid-state electron multiplying structure based on the Impact Ionization phenomenon in silicon. Here we review this technology, and evaluate the first electron multiplying CCD camera, in particular using it to image weak emissions form microtitre plates. A theoretical model was constructed to predict S/N and Z-factor performances, which were compared to actual measurements, verifying that a greater than one order of magnitude improvement can be achieved over conventional CCDs. The demonstrations of remarkable sensitivity enhancement presented here are discussed in terms of the EMCCD camera's suitability for use in life sciences applications such as High-Throughput Screening (HTS), and other approaches requiring ultrasensitive detection of biomolecules, including Single Molecule Detection.

Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

Four different algorithms are presented which generate potentially well biding peptides against specific mono- clonal antibodies. The input data for these algorithms comes form random peptide array screening experiments, which results in a binding strength for a few thousand peptides against the antibody. The first algorithm identifies short motifs of amino acids which occur more frequently among the best binding peptides than among the worst binding ones. The second algorithm differs from this algorithm in the sense that it searches for amino acids in the best binding peptides, regardless of the order of these amino acids. The fourth algorithm replaces all amino acids by a hydrophilicity value and starts to search for clusters in the profiles of the best measured results. Results obtained from experimental data show that the algorithms are able to generate peptides which obtain a resemblance with the peptides which are known to bind reasonably well against these antibodies. The information gained from these algorithms is useful for the design of subsequent experiments aimed at further optimization of the best binding peptides found during the peptide screening experiment.

High-Throughput Screening (HTS) data in its entirety is a valuable raw material for the drug-discovery process. It provides the most compete information about the biological activity of a company's compounds. However, its quantity, complexity and heterogeneity require novel, sophisticated approaches in data analysis. At GeneData, we are developing methods for large-scale, synoptical mining of screening data in a five-step analysis: (1) Quality Assurance: Checking data for experimental artifacts and eliminating low quality data. (2) Biological Profiling: Clustering and ranking of compounds based on their biological activity, taking into account specific characteristics of HTS data. (3) Rule-based Classification: Applying user-defined rules to biological and chemical properties, and providing hypotheses on the biological mode-of-action of compounds. (4) Joint Biological-Chemical Analysis: Associating chemical compound data to HTS data, providing hypotheses for structure- activity relationships. (5) integration with Genomic and Gene Expression Data: Linking into other components of GeneData's bioinformatics platform, and assessing the compounds' modes-of-action, toxicity, and metabolic properties. These analyses address issues that are crucial for a correct interpretation and full exploitation of screening data. They lead to a sound rating of assays and compounds at an early state of the lead-finding process.

G protein-coupled receptors (GPCRs) are historically the richest targets for drug discovery, accounting for nearly 60 percent of prescription drugs. The ligands and functions of only 200 out of possibly 1000 GPCRs are known. Screening methods that directly and accurately measure GPCR activation and inhibition are required to identify ligands for orphan receptors and cultivate superior drugs for known GPCRs. Norak Biosciences utilizes the redistribution of a fluorescently-labeled protein, arrestin, as a novel screen for monitoring GPCR activation. In contrast to the present methods of analyzing GPCR function, the power of the Transfluor technology is in its simplicity, large signal to noise ratio, and applicability to all GPCRs. Here, we demonstrate that the Transfluor technology can be automated and quantitated on high throughput image analysis systems. Cells transfected with an arrestin-green fluorescent protein conjugate and the neurokinin-1 GPCR were seeded on 96-well plates. Activation of the NK-1 receptor with Substance P induced translocation of arrestin-GFP from the cytosol to the receptor. Image quantitation of the arrestin-GFP translocation was used to generate dose dependent curves. These results reveal that the Transfluor technology combined with an image analysis system forms a universal platform capable of measuring ligand-receptor interactions for all GPCRs.

Assay miniaturization and the implementation of high-density 1536 micro-well screening increases the speed and efficiency of screening and lead discovery. To serve this need, a platform of miniaturizable assay technologies has been assembled for specific biological targets. This platform will enable initiation and completion of uHTS screens in a straightforward and expeditious manner. For kinases, we have examined assays using several technologies including DELFIA, HTR-FRET, FP, EFC, and FMAT. This presentation compares these technologies for the measurement of typical tyrosine kinase activity in 1536-well format. Quality parameters such as assay reproducibility, signal: background ratio, Z factor, and assay sensitivity were calculated and compared. Additionally, the relative merits of each of these technologies were assessed in terms of assay miniaturization, ease of development, ultimate screening capability, efficiency, and cost.

We have developed a novel microarray technology for performing very large numbers of biochemical, chemical and cell-based nanoliter volume synthesis, storage and screening operations in a massively parallel manner. The Living Chip is an array of precisely machined through-holes retaining nanoliters of fluid by capillary action. Sample loading, washing and recovery are operations that can be performed manually or with simple automation. Mixing between co- registered through-holes is achieved by stacking two or more precision aligned arrays and optical assay read-outs are in parallel with a CCD imaging system. An automated picker system transfers hits into lower density microtiter plates for further analysis. We will present result demonstrating massively parallel implementation of both homogeneous and inhomogeneous fluidic and cell-based assay systems and applications of the chip for drug compound library storage and management.

The process of drug discovery can be accelerated by increasing the information content of bioassays and by employing assay platforms that are amenable to high throughput screening techniques. In this paper, we demonstrate how the combination of soft lithography with controlled surface chemistry achieves these goals in a wide spectrum of bioassays. A number of soft lithographic methods can be used to generate micro-structures for the purposes of increasing assay density, diversity of test conditions and improving assay detection qualities. In addition, soft lithography, combined with specific surface chemistry modification procedures and protein engineering, may be used to control the localized molecular and biological properties of substrates, thereby enabling the development of new types of bioassays. The developed methodologies are simple, easily implemented, and lend themselves well to automation. Experimental results and prototypes are presented to illustrate the capabilities of these new techniques. For example, soft lithography and surface chemistry are employed for chemically patterning substrates, stenciling biological entities onto substrates and confining solutions. As a result, information-rich, highdensity bioassays can be obtained where biological targets, surface properties and medium solutions are carefully determined and controlled.

OmniPlex Technology is a new approach to genome amplification and targeted analysis. Initially the entire genome is reformatted into small, amplifiable molecules called Plexisomes, which represent the entire genome as an OmniPlex Library. The whole genome can be amplified en masse using universal primers; using locus-specific primers, regions as large as 50 kb can be amplified. Amplified Plexisomes can be analyzed using conventional methods such as capillary sequencing and microarray hybridization. The advantages to using OmniPlex as the 'front-end' for conventional analytical instruments are that a) the initial copy number of the analytes can be increased to achieve better signal-to-noire ratio, b) only a single priming site is used and c) up to 20 times fewer biochemical reactions and oligonucleotides are necessary to amplify a large region, compared to conventional PCR. These factors make OmniPlex more flexible, faster, and less expensive than conventional technologies. OmniPlex has been applied to targeted sequencing of human, animal, plant, and microorganism genomes. In addition, OmniPlex is inherently able to haplotype large regions of human DNA to accelerate target discovery and pharmacogenomics. OmniPlex will be a key tool for delivery of improved crops and livestock, new pharmaceutical products, and personalized medicine.

Numerous regulatory documents and a wide variety of books (1-4), papers and technical standards provide guidelines for method validation. Regardless of the numerous sources providing general direction to the method developer, “validation” is an extremely complex regulatory issue that is not easily defined. Proving the specificity, linearity and range of NIR methods provides unique challenges relative to more traditional analytical techniques. Various approaches for proving the validity of these performance parameters exist.