Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

Molecular classification of tumors holds great potential for cancer research, diagnosis, and treatment. In this study, we apply a novel classification technique to cDNA microarray data for discriminating between three subtypes of malignant lymphoma: CD5+ diffuse large B-cell lymphoma, CD5- diffuse large B-cell lymphoma, and mantle cell lymphoma. The proposed technique combines the k-Nearest Neighbor (k-NN) algorithm with optimized data quantization. The feature genes on which the classification is based are selected by ranking them according to their separability criteria computed by taking into account between-class and within-class scatter. The classification errors, estimated using cross-validation, are significantly lower than those produced by classical variants of the k-NN algorithm. Multidimensional scaling and hierarchical clustering dendrograms are used to visualize the separation of the three subtypes of lymphoma.

The desire to obtain more biologically relevant data is expanding the use of cell-based assays in drug discovery. These assays are performed and analyzed in ever more sophisticated ways (e.g. high content screening) that allow the collection of multiparametric information about cells affected by the screened compounds. The driver for these developments is the desire to increase data quality and density and reduce the use of valuable reagents and time. Here we describe an approach that adds a new dimension to the data quality/quantity mix by simultaneously analyzing several cell types in the same microplate well. The system is based on the use of encoded carriers (CellCards) that permit the reading and analysis of cellular responses, and at the same time allow decoding and the attribution of these responses to the appropriate cell line. CellCards are rectangular particles with an expandable color barcode and a transparent section upon which cells can be grown and imaged for cellular readout. Before performing the assay, each cell line is grown on a different class of CellCards. CellCards, with attached cells, are mixed and dispensed into a microtiter plate where the assay is performed. Next the plates are imaged, decoded and the cells associated with each CellCard class are analyzed. Multiplexing cell lines allows assay controls and data normalization within each well, reducing well-to-well variability. It also allows the simultaneous interrogation of multiple targets and thus concurrent potency and selectivity screening. This may significantly reduce the time required to take a compound from primary screening into the clinic.

Background: Slide based cytometry (SBC) is a technology for the rapid stoichiometric analysis of cells fixed to surfaces. Its applications are highly versatile and ranges from the clinics to high throughput drug discovery. SBC is realized in different instruments such as the Laser Scanning Cytometer (LSC) and Scanning Fluorescent Microscope (SFM) and the novel inverted microscope based iCyte image cytometer (Compucyte Corp.). Methods: Fluorochrome labeled specimens were immobilized on microscopic slides. They were placed on a conventional fluorescence microscope and analyzed by photomultiplayers or digital camera. Data comparable to flow cytometry were generated. In addition, each individual event could be visualized. Applications: The major advantage of instruments is the combination of two features: a) the minimal sample volume needed, and b) the connection of fluorescence data and morphological information. Rare cells were detected, frequency of apoptosis by myricetin formaldehyde and H₂O₂ mixtures was determined;. Conclusion: LSC, SFM and the novel iCyte have a wide spectrum of applicability in SBC and can be introduced as a standard technology for multiple settings. In addition, the iCyte and SFM instrument is suited for high throughput screening by automation and may be in future adapted to telepathology due to their high quality images. (This study was supported by the IZKF-Leipzig, Germany and T 034245 OTKA, Hungary)

Spinning-disk self-referencing laser interferometers are being developed as high-speed high-sensitivity platforms for immunoassay and proteomics applications. Their compact disc (CD) formats have the potential for ultra-high-throughput multianalyte assays as well as for binding kinetics and quantitative analysis. Self-referencing interferometers are immune to mechanical variations, enabling interferometric sensitivities and speeds that are several orders of magnitude larger than for their counterpart fluorometric techniques. This paper defines for the first time three classes of the BioCD that differ in their method of self-referencing and reviews their relative merits and sensitivities. Each uses a near-field probe with far-field detection. The three classes are: microdiffraction, adaptive optical, and photonic cavity.

The adsorption of five proteins with very different molecular characteristics, i.e. α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin, has been characterized using quantitative fluorescence measurements and atomic force microscopy. It has been found that the 'combinatorial' nature of the micro/nano-channels surface allows for the increased adsorption of molecularly different proteins, comparing with the adsorption on flat surfaces. This amplification increases for proteins with lower molecular surface that can capitalize better on the newly created surface and nano-environments. Importantly, the adsorption on micro/nano-fabricated structures appears to be less dependent on the local molecular descriptors, i.e. hydrophobicity and charges, due to the combinatorialization of the nano-areas presented to the proteins. The amplification of adsorption is important, ranging from 3- to 10-fold, with a higher amplification for smaller, globular proteins.

We demonstrate a novel method for the control of small droplets using laser-based heating. Temperature dependent interfacial surface tensions were the primary force used to move droplets. With this approach, ~1.7 μL to 14 pL droplets were moved on a bare, unmodified polystyrene surface, at speeds of up to 3 mm/s. Upon contact, droplets spontaneously fused and rapidly mixed within 33 ms. We performed an optical absorption-based protein assay using horseradish peroxidase and a chromogenic substrate (ABTS), and readily detected as little as ~125 attomoles of reacting enzyme.

We describe real-time detection of antigen/antibody binding using a high-speed label-free interferometric detection technique. The sensor, called the BioCD, consists of microfabricated gold interferometric structures on 2” dielectric laser mirror substrates that spin at rates up to 6000 rpm. The interferometric microdiffraction elements operate in the linear sensitivity regime of the interferometer. Antibodies or proteins are immobilized on the gold interferometric structures through an intermediate thiol layer. The molecules are immobilized by application of reagents or samples to the disk while it is spinning. The centrifugal force distributes the sample over the sensor surface, causing a change in the optical phase of the interferometric element, which is detected in real-time using lock-in detection with small detection bandwidth. The thiolated BioCD is spun at 1500 rpm and anti-mouse IgG, rabbit IgG and mouse IgG are delivered in succession to the sensor surface with potash buffered saline (PBS) wash cycles interspersed between each exposure to remove excess unbound proteins. The layer of anti-mouse IgG binds to the thiolated gold sensor elements which later bind specific mouse IgG. We have observed a 10% change in the interferometric signal when mouse IgG binds to an immobilized layer of anti-mouse IgG. No significant non-specific binding of rabbit IgG was detected. The sensitivity and throughput of this sensor will be discussed. An advantage of this new approach, relative to previous work in which the disk was incubated with antigen off-line, is the real-time detection of antigen binding, which could be valuable for simultaneous high-speed screening of a large number of protein interactions.

In the development of HTS as a central paradigm of drug discovery, fluorescent reporter molecules have generally been adopted as the favored signal transducer. Nevertheless, luminescence has maintained a prominent position among certain methodologies, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a broader range of applications due to their sensitivity, extensive linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by development several new assay designs for diverse targets such as kinases, cytochrome p450's, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for drug discovery, highlighting new detection capabilities brought about by engineering luciferase enzymes and substrates. In reporter gene applications, modified luciferases have provided greatly improved expression efficiency in mammalian cells, improved responsiveness to changes of transcriptional rate, and increased the magnitude of the reporter response. Highly stabilized luciferase mutants have enabled new assays strategies for high-throughput screening based on detection of ATP and luciferin. Assays based on ATP support rapid analysis of cell metabolism and enzymatic processes coupled to ATP hydrolysis. Although luciferin is found natively only in luminous beetles, coupled assays have been designed using modified forms of luciferin requiring the action of second enzyme to yield luminescence. Due to the very low inherent background and protection of the photon-emitter afforded by the enzyme, bioluminescent assays often outperform the analogous fluorescent assays for analyses performed in multiwell plates.

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology’s Invader methods.

We present a fluorescence/luminescence imaging system for use in high-throughput screening of samples in microplates. High efficiency imaging of microplates is an optical challenge usually involving performance compromises. Conventional microplate imagers use large refractive systems. These systems typically have many large lenses and suffer from their disadvantages such as low light transmission efficiency, field shading (low uniformity), and weight. Our optical design enables simultaneous detection of light from all of the samples in a microplate with high collection efficiency, high transmission efficiency, low chromatic aberration, high uniformity, and sub-well (submillimeter) resolution all in a relatively small package. Our optical system includes a primary mirror and an on-axis secondary mirror with a central aperture (a reverse-conjugate Schwarzchild arrangement) along with a small set of refractive field correctors. The field correctors compensate for the aberrations induced by the wide field (108×72 mm) without resorting to aspheric mirrors. The prototype of this design met our goals of high transmission, high uniformity and low crosstalk.

Gene Array technology has allowed for the study of gene binding by creating thousands of potential binding sites on a single device. A limitation of the current technology is that the effects of the gene and the gene-derived proteins cannot be studied in situ the same way, thousand site cell arrays are not readily available. We propose a new device structure to study the effects of gene modification on cells. This new array technology uses electroporation to target specific areas within a cell culture for transfection of genes. Electroporation arrays will allow high throughput analysis of gene effects on a given cell's response to a stress or a genes ability to restore normal cell function in disease modeling cells. Fluorescent imaging of dye labeled indicator molecules or cell viability will provide results indicating the most effective genes. The electroporation array consists of a microelectronic circuit, ancillary electronics, protecting electrode surface for cell culturing and a perfusion system for gene or drug delivery. The advantages of the current device are that there are 3200 sites for electroporation, all or any subsets of the electrodes can be activated. The cells are held in place by the electrode material. This technology could also be applied to high throughput screening of cell impermeant drugs.

Microarray has revolutionized the study of molecular biology, especially the application in clinical diagnostics. When used in clinical diagnostics, microarray has to meet a high degree of reproducibility, reliability and quality in order to become a standard tool. Repeatability and reproducibility are essential for providing the best data and process control. The real challenge for microarray is, however, how to produce consistent and reliable data. The variance of microarray data is contributed by the quality of sample source, the quality of glass substrates, hybridization, and probe labeling and spotting. The quality of coated glass substrates is one of the main factors. This paper is focusing on discussing how to optimize coating conditions to improve the slide quality, consequently improve the data quality such as sensitivity and data reliability.

We developed an optical oblique-incidence reflectivity difference (OI-RD) scanning microscope for imaging microarrays of label-free protein and DNA. Such a microscope complements currently widely used fluorescence-based optical microscopes by offering the capability to detect biochemical activities of DNA and protein molecules without the influence of fluorescent-labeling molecules. The specific activity and function of protein molecules are particularly subject to binding of small or large foreign molecules either directly through conformational change in the protein molecule itself or indirectly through properties of the attached molecules. We show that an OI-RD microscope can be used to: 1) detect binding reactions on microarrays without labeling, 2) quantitatively measure the optical properties of microarray spots, and 3) detect microarrays submerged in solution (which will enable OI-RD to monitor reaction kinetics on microarrays in reactive solutions). Furthermore, using both OI-RD and fluorescence images of an immunoglobulin-G (IgG) protein microarray, we observed that labeled and unlabeled IgG molecules deposited on the microarray substrate exhibit different wetting behaviors, and a mixture of the two tends to segregate into labeled and unlabeled regions. This illustrates potentially undesirable effects of fluorescent-labeling agents on protein properties that are of interest.

Efficient hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for gene expression analysis. Recently studies have been published to assess oligonucleotide (55-70 bases) performance on glass-slide microarrays and stress advantages over the cDNA arrays. Importantly the oligo arrays eliminate possible failure in PCR amplifications and attain sequence optimization. In the present study, we have used 60mer oligo microarrays to investigate the effect of target (immobilized on the glass slides) and probe concentrations and possible probe interactions on hybridization. Scanner calibration slides (manufactured by Full Moon BioSystems) were used to concert the fluorescence signals into fluorophore per μm² in order to eliminate possible variation from scanner. The retention of the target was determined based on mock hybridization using Cy3-labeled oligonucleotide. We found that hybridization signals fell within the linear response range when the target concentration (printing solution) was equal or less than 2.5 μM. With fixed target concentrations, there is a non-linear relationship between the probe concentration and the hybridization signal. Dual-probe hybridization measurements suggest that hybridization of probes is not ideally independent. In this study, hybridization signal from Cy5 is consistently lower than that from Cy3.

We report a label-free, highly sensitive biosensor using a vertical cavity surface emitting laser (VCSEL) based measurement system for the detection and monitoring of biomolecular interactions. The sensor system consists of a VCSEL, a plastic guided mode resonant (GMR) filter, and two pin detectors. The system has several advantages such as extreme compactness, high sensitivity, high throughput, fast measurements, low power consumption, low cost, and the potential to become portable. Experimentally, the biosensor system has shown to be highly sensitive to the surface modifications due to molecular bindings, with the ability to detect the thickness variations <10Å and refractive index variations <0.005. The biosensor also has demonstrated its high sensitivity for the detection of antibody-antigen proteins bindings, with the mouse IgG concentration as low as 1pg/ml (6.7 femto-Molar), and its ability for measuring both static and dynamic interactions among proteins.