Illumina is developing a BeadArray^TM technology that supports SNP genotyping, mRNA expression analysis and protein expression analysis on the same platform. We use fiber-optic bundles with a density of approximately 40,000 fibers/mm². At hte end of each fiber, a derivatized silica bead forms an array element for reading out a genotyping or expression assay data point. Each bead contains oligonucleotide probes that hybridize with high specificity to complementary sequences in a complex nucleic acid mixture. We derivatize the beads in bulk, pool them to form a quality-controlled source of microarray elements, and allow them to assemble spontaneously into pits etched into the end of each optical fiber bundle. We load our fiber bundles, containing 49,777 fibers, with up to 1520 different bead types. The presence of many beads of each type greatly improves the accuracy of each assay. As the final step in our manufacturing process, we decode the identity of each bead by a series of rapid hybridizations with fluroescent oligos. Decoding accuracy and the number of beads of each type is recorded for each array. Decoding also serves as a quality control procedure for the performance of each element in the array. To facilitate high-throughput analysis of many samples, the fiber bundles are arranged in an array matrix (Sentrix^TM arrays). Using a 96-bundle array matrix, up to 1520 assays can be performed on each of 96 samples simultaneously for a total of 145,920 assays. Using a 384-bundle array matrix, up to 583,680 assays can be performed simultaneously. The BeadArray platform is the highest density microarray in commercial use, requiring development of a high-performance array scanner. To meet this need, we developed the Sherlock^TM system, a laser-scanning confocal imaging system that automatically scans all 96 bundles of an array matrix at variable resolution down to 0.8 micron. The system scans with both 532 and 635 nm lasers simultaneously, collecting two fluorescence images. The optical train is designed around a telecentric, flat field, macro scan lens with a field of view of 2 mm. Our BeadArray platform is adaptable to many different assays. In our genotyping services lab, we automated the development and production of highly multiplexed SNP genotyping assays. Each SNP call is made automatically and assigned a quality score based on objective measures of allele clustering across multiple samples. The quality score correlates directly with genotyping accuracy. With a small number of robots and thermal cyclers, and a team of 5 people, we have the capacity to perform over 1 million genotypes per day. The system is modular so that scale-up is limited only by demand. The system has the capacity, versatility, and cost structure to meet the needs of large-scale genomic analysis.

The latest self-encoding resin bead is a novel technology for solid phase synthesis combinatorial library screening. A new encode-positional deconvolution strategy which was based on that technology been illustrated compared with positional scanning and iterative strategies. The self-encoding resin beads technology provides an efficient method for improving the high-throughput screening of combinatorial library.

Photothermal deflection technique is a very sensitive mean to measure optical absorbance. This study is aimed at evaluating its potentiality in order to detect hybridation and to monitor quality control. The principle of the technique relies on the fact that nucleic acids present the property to absorb light between 220 nm to 280 nm. A first theoretical approach based on solving light and thermal equations demonstrates the ability of photothermal deflection to detect hybridisation. This point is checked through several experimentations with oligonucleotid with 32 mers targets lengths. An important point to stres is the specificity of the signals obtained and the ability of automation of the reading with image processing algorithms. At last we focus our attention on the ability of this technique to in-situ synthesis process. Our experimental study shows the ability of this characterisation with a detection sensitivity of one base.

A novel surface plasmon resonance (SPR) imaging system based on modified Mach-Zehnder phase-shifting interferometry (PSI) measures the spatial phase variation of a resonantly reflected light in biomolecular interaction. In this technique, the SPR DNA microarray can diagnose the target DNA without additional labeling in real-time biomolecular interaction analysis (BIA). Owing to the feasible and swift measurements, the SPR microarray with this SPR imaging system can be extensively applied to the nonspecific adsorption of protein, the membrane-protein interaction, receptor-ligand interaction, DNA hybridization. The detection limitation of the SPR PSI imaging system is improved to about 1 pg/mm² surface coverage of biomaterial for each individual spot over that of the conventional SPR imaging system that observe the intensity variation of a resonantly reflected light. The SPR PSI imaging system and its SPR microarray can provide the capability to analyze DNA hybridization or protein interaction in real-time, with high resolution, and at high-throughout screening rates.

Perennial ryegrass (Lolium perenne) is a major forage grass of temperate pastures. A genomics program has been undertaken generating over 52,000 expressed sequence tags (ESTs). Cluster analysis of the ESTs identified approximately 14,600 ryegrass unigenes. In this report, we described the application of ryegrass unigene cDNAs to produce ryegrass 15K microarray. Fifteen microarray hybridisations were performed with labeled total RNA isolated from a variety of plant organs and developmental stages. In a proof of concept, gene expression profiling of ryegrass ESTs using the 15K unigene microarrays has been established using several known genes and two cluster analysis approaches (parallel coordinate planes plot and hierarchical clustering). The expression profile of the known genes (e.g. rubisco and invertase) corresponds well with published data. The microarray expression profile of a ryegrass putative root specific kinase gene was also verified with Northern blotting. This combination of DNA microarray hybridisations and cluster analysis can be applied as a tool for the identification of novel sequences of unknown function.

Investigation of protein-polymeric surface interaction requires reliable practical techniques for evaluation of the efficiency of protein immobilization. In this study the efficiency of protein immobilization was evaluated using three different techniques: (1) protein-binding assay with fluorescent detection and (2) quantification, and (3) atomic force microscopy. This approach enables us to rapidly analyse the adsorption properties of different proteins. The comparative physico-chemical adsorption of α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin in the micro-wells fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm) deposited on a Poly(methyl ethacrylate) film has been studied. Correlations were observed between the quantitative and qualitative differences depending on both protein and polymeric surface hydrophobicity.

Interferometers can detect optical path changes down to a billionth-lambda at the half intensity point at quadrature, (defined when the signal and reference waves are out of phase by ninety degrees). We have fabricated interferometric microstructures on silicon all operating at quadrature. The ultimate capability of this approach is the fabrication of over a billion interferometric biosensors on a single spinning disk having the capacity for mega-samples per second sampling speed. As an initial proof of principle of this technique, we have detected the presence of immobilized anti-mouse IgG and the specific binding of mouse IgG at a sampling rate of 100kiloSamp/sec, while non-specific binding observed was low. We will demonstrate that this technique provides a label-free method that may rapidly screen thousands of proteins per assay.

A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10^-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

In previous publications and presentations we have described our construction of a laboratory-on-a-chip based on nanoliter capacity wells etched in silicon. We have described methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the production or consumption of NADH in enzyme-catalyzed reactions such as those found in the glycolytic pathway of yeast. In this paper we describe the use of light sensors (photodiodes) in each well to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence in 100 μM and for ATP/luciferase bioluminescence is 2.4 μM.

We recently described a technique to fabricate shallow (< 50 nm) microstructures on PMMA surface for use in multianalyte protein micro-assay based on the ablation of a top thin gold layer using pulsed nitrogen laser (337 nm). In the present study, AFM has been used to investigate the surface characteristics and to provide physical insights into the formation of these complex microstructures. It has been shown that lateral diffusion of the heat generated during the gold ablation extended to ca. 3 μm on either side of the laser focal spot (ca. 5μmm wide), effectively ablated the gold layer and created shallow regions of ca. 20 nm. The heat also created a depression (ca. 5 μm wide) in the polymer region at the laser spot, and a hump, that increased in height with laser dose, at the center of the depression. It is suggested that volume shrinkage caused by stress relaxation and material redistribution, and volume expansion caused by fragmentation of the polymer are responsible mechanisms. Chemical changes also occurred resulting in the middle zone of the microstructure, which corresponds to the central hump, being hydrophobic, whereas the outer zone was hydrophilic. It is suggested that degraded hydrophilic products may be present in the outer zone, whereas the middle zone may contain smaller hydrophobic fragments due to more advanced fragmentation. The variation in the morphology and surface chemistry in the shallow microstructures effectively 'combinatorialize' the surface properties of the microstructures, thus facilitating the patterning of different proteins.

We have successfully demonstrated the development of a compact and cost-effective parallel multi-channel capillary electrophoresis system for bio-molecules analysis. The automated process includes a buffer/gel replenishment mechanism, high voltage control of fluidics and an automated sample tray transport capability. The bio-separation/analysis occurs in a disposable cartridge containing multi-column capillaries with integrated excitation optical fibers, detection micro-optics and a buffer reservoir common to all separation channels. Tests of this fully integrated system indicate, that large quantities of biological samples can be analyzed automatically in a short period with highly sensitive fluorescence detection.

Two different methods of automated high throughput purification of genomic DNA from human whole blood in 96 well plates are described. One method uses MagneSil^TM paramagnetic particles to purify a maximal amount of the DNA present in the sample. Another method, the Magnesil^TM ONE system, allows for the purification of a predetermined amount of DNA from human whole blood. Protocols for the purification of 100 ng or, alternatively 1 ug, of human genomic DNA from whole blood using MagneSil^TM paramagnetic particles and a Beckman BioMek^TM FX robot are described. With the maximal yield purification system, typical DNA yields fall in the range of 4-9 ug of DNA from 200ul of human whole blood, depending upon the white cell content of donor sample. For situations where DNA achiving is desired, or when the number of downstream sample applications is not clearly defined (e.g. multiple SNP analyses) the maximal yield method is usually preferred. However, in situations with a defined downstream application (e.g. criminal databasing or use of a defined set of amplifications) where purifying DNA in a narrow concentrate range streamlines the high throughput purification and analysis process, the automated MagneSil^TM ONE purification system is the method of choice. DNA from either method is suitable for applications such as PCR, STR, READIT^TM SNP analysis, and multiplexed PCR systems such as Promega's Y-chromosome deletion detection system.

We developed a simulation model of an integrated CMOS-based imaging platform for use with bioluminescent DNA microarrays. We formulate the complete kinetic model of ATP based assays and luciferase label-based assays. The model first calculates the number of photons generated per unit time, i.e., photon flux, based upon the kinetics of the light generation process of luminescence probes. The photon flux coupled with the system geometry is then used to calculate the number of photons incident on the photodetector plane. Subsequently the characteristics of the imaging array including the photodetector spectral response, its dark current density, and the sensor conversion gain are incorporated. The model also takes into account different noise sources including shot noise, reset noise, readout noise and fixed pattern noise. Finally, signal processing algorithms are applied to the image to enhance detection reliability and hence increase the overall system throughput. We will present simulations and preliminary experimental results.

Fluorescence Correlation Spectroscopy (FCS) may be used to assay the binding of drug-like ligands to signaling proteins and other bio-particles. For High Throughput Screening (HTS), a competitive format is typically used in which binding of an unlabeled compound results in displacement of a fluorescently labeled ligand. Unweighted curve-fitting of the normalized autocorrelation function (ACF) to a two-diffusion-component model can resolve the fractions of free and bound ligand if the diffusion rates differ sufficiently and if the experimentally estimated ACF has adequate precision. However, for HTS (and also for intracellular FCS studies) it is desirable to minimize the experimental data collection time. In this case, the precision of the ACF is limited and it becomes important to account for the statistical features of the ACF estimate when designing an assay. The errors at different points in the estimated ACF are correlated and hence least-squares fitting methods are not strictly statistically rigorous. We compare different methods for estimating and curve-fitting the ACF from the raw data of short duration FCS measurements. The methods are applied to data from experiments to assay binding of Alexa-488-labeled Bak peptide with Bcl-x_L, which is an intracellular protein that acts to protect against programmed cell death. We present results from a detailed Monte Carlo simulation of the experiment, which is useful for validating short-duration assay capabilities. We also discuss the measurement of changes in steady state fluorescence anisotropy due to restricted rotational diffusion upon binding, which provides a complementary assay method.

The goal of this study is to test the feasiblity of directly using molecular descriptor data to generate clusters of similar molecules. We have developed an approach that utilizes the "most orthogonal" molecular descriptor variables as a basis set for clustering. In this study we have specifically utilized normal skin tissue and melanoma cancer data derived via Fourier transform infrared (FTIR) spectroscopy to generate these clusters, but the approach presented should be applicable to any other molecular descriptor or response data. Using the three most orthogonal FTIR frequencies as a basis set for cluster analysis, normal skin and melanoma tumors' clusters were resolved and localized in the three-dimensional variable/frequency space. Such clusters can be used to rapidly identify molecules with similar structures, and biological activity given their physico-chemical descriptors or molecular response data. This study also points out possible fallacies when inspecting clusters and how they can be avoided.

Methods for DNA testing are abundant, however, there is still a need for lower cost, higher-throughput genotyping. Much emphasis has been placed on short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs). Another important polymorphism is the diallelic short insertion/deletion (indel). Our laboratory is using a continuous reel of 384 well arrays on polypropylene tape to genotype indel polymorphisms. The reel of arrays allows low cost automation, and an opportunity to decrease reaction volumes. The diallelic indel is typed by tagging allele-specific PCR primers with a FAM or JOE molecular beacon uniprimer. Our most recent array pattern contains wells that hold a maximum of 1.1 microliters with reaction volumes of 800 nanoliters. Because micro array tape (microtape) is unique, commercial equipment is not yet available. A series of instruments were developed in-house to handle the tape. A pipetting instrument was developed to deliver the DNA samples or other reagents. A solenoid micro-valve aspirating and jetting unit was developed for dispensing a common reaction mix. The arrays are sealed with commercially available microtiter plate seal material prior to polymerase chain reaction (PCR) within a custom built waterbath thermal cycler. The arrays are scanned using an epi-fluorescence detection unit designed to read FAM and JOE fluorescent dyes. The detector uses an argon ion laser for excitation and two photomultiplier tubes (PMTs) for detection. The resulting images are processed using custom software.

In this paper, we propose a new nonlinear matching measure for automatic analysis of the on-off type DNA microarray images in which the hybridized spots are detected by the template matching method. The targeting spots of HPV DNA chips are designed for genotyping the human papilloma virus(HPV). The proposed measure is obtained by binarythresholding over the whole template region and taking the number of white pixels inside the spotted area. This measure is evaluated in terms of the accuracy of the estimated marker location to show better performance than the normalized covariance.

In this paper we will describe a system designed to combine optical tweezers and laser scalpels with confocal as well as epi-fluorescence microscopy. A continuos wave Nd:YVO₄ laser is used to produce a dual optical tweezers, where each trap can be individually controlled. A second optical tweezers setup is based on a tunable titanium sapphire laser, which allows us to adjust the wavelength to minimize the damage to the cell under investigation. A pulsed nitrogen laser working at 337 nm forms a laser scalpel. The tweezers and scalpels are both incorporated in an inverted microscope equipped with epi-fluorescence and confocal imaging capabilities. In order to further control the sample we have developed a technique to tailor make the environment closest to the studied objects. Micrometer-sized structures such as channels and reservoirs have been produced in rubber silicon using lithographic methods. In combination with a micro-manipulator, our system can be used to extract single cells from a population of billions for further studies or growth.