Journal of Laboratory Automation

Evaluating changes in gene expression is essential when identifying and validating new drug targets, however, the methods used to measure transcription are laborious, time-consuming and expensive (e.g., RT-PCR, microarray or northern blot). High Performance Signal Amplification (HPSA™) gene expression assays quantitate particular mRNA targets directly in cell lysate samples using DNA probe hybridization and fluorescent signal amplification. The assay format eliminates the need for RNA purification prior to testing and does not require RT-PCR amplification. The HPSA™ protocol involves three steps carried out at 37°C in 96- or 384-well plates, making the technique amenable to automation. Cellular mRNA levels are quantitated relative to a standard curve consisting of purified in vitro RNA transcripts derived from cDNA clones. Assay sensitivity is in the low attomole range and can detect mRNA expressed at twenty copies per cell. A number of HPSA™ assays have been developed for cytokine, housekeeping and cytochrome P450 messages. Gene induction profiles were monitored in cell lines or peripheral blood mononuclear cells (PBMC) treated with activators such as phorbol 12-myristate-13-acetate (PMA), ionomycin or phytohemagglutinin (PHA). Recent validation studies demonstrated IL-9 mRNA induction in primary cultures of T-cells treated with PMA and anti-CD3. Similar testing with PBMCs showed significant IL-13 mRNA induction after 48 hours of treatment with PMA, thymosin and staphylococcal enterotoxin. Cytokine STATE® panels are being constructed according to functional categories such as innate or adaptive immunity to better characterize changes in transcription patterns during an immune response. The HPSA™ gene expression assays offer a rapid and convenient alternative to more cumbersome, expensive methods.

IQ™ Technology is a homogeneous, universal detection platform designed specifically for high-throughput screening of kinases and phosphatases. The technology has been tested in 96- to 384- to 1536-well microplate formats and is ideally suited for automated drug discovery screening systems. IQ™ Technology is a direct, non-competitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of fluorophore-labeled peptide substrates. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide, because the iron-containing compound quenches the fluorescent emission exclusively on the phosphorylated form of the fluorophore-labeled peptide. The IQ™ Technology provides a universal method that can be used with any peptide sequence.

Traditional organic synthesis is often cumbersome and time consuming. Significant effort by manufacturers of automated systems has been directed at increasing the speed, efficiency, and consistency of performing chemical reactions. To date, the numbers of integrated systems that combine all the relevant steps of compound preparation are few.

Using the five automated systems in tandem can successfully streamline research and development of potential drug candidates. The features of these workstations offer a unique approach for supporting the convenient synthesis and workup of diverse compounds without compromising reagent types or conditions used for synthesis.

This poster details an example of the automated drug discovery approach to high throughput organic synthesis using the solution phase synthesis of a series of 1-indanones as building blocks and subsequent reductive amination reactions to generate a diverse group of amines. The purpose of each automated system is outlined along with its role in generating the small library as an illustration of the importance of time saving devices in laboratories.

Pharmaceutical and biotechnology research requires instrumentation to be both functional and versatile. In the HTS and Drug Discovery environments, microplate-based assays are developed to make determinations on large numbers of samples. Regardless of the assay protocol, the end result is the measurement by some sort of detection device. Towards that end, Bio-Tek has developed the Synergy™ HT Multi-Detection Microplate Reader. Synergy HT utilizes two independent sets of optics to provide uncompromised performance. For absorbance measurements, there is a xenon-flash lamp with a monochromator for wavelength selection and photodiode detection. This allows the selection of any wavelength for endpoint or kinetic measures from 200 nm to 999 nm in 1 nm increments, as well as spectral scans. Traditional visible wavelength fluorescence measurements are made using a tungsten-halogen lamp with interference filters (excitation and emission) for wavelength selection and photomultiplier (PMT) detection. Glow luminescence measurements are also easily accomplished in Synergy H T. If time-resolved or UV excitation fluorescence measurements are required, Synergy HT automatically integrates the xenon-flash-monochromator excitation with the interference emission filter and PMT detection. Typical applications include antibody-antigen binding, receptor-liquid binding, ELISA, nucleic acid quantitation using fluorescent dyes or direct UV analysis. Synergy HT is capable of reading any plate format up to 384-well plates and provides temperature control up to 50°C and shaking as standard features. It is compact (16″ W × 15″ D × 10″ H) and robotics-compatible through the OLE functionality of the KC4™ data reduction software that is bundled with the instrument. An upgrade to CFR 21 Part 11 compliant software is also available.

Genome Therapeutics Corp. has implemented a unique maintenance approach for their GenomeVision™ Services 24-by-7 high-throughput Sequencing platform that ensures optimal performance and minimum downtime. A network-enabled software program automatically coordinates and tracks all maintenance tasks, and notifies responsible personnel by e-mail of their upcoming maintenance responsibilities. Production personnel perform all internal scheduled instrumentation maintenance, equipment vendors perform purchased service contracts, and a small in-house group responds to emergency situations. Personnel log completed maintenance tasks and request emergency service by means of a network-based interface that results in rapid response of appropriate in-house personnel or outside service organizations. The proprietary software program tracks all maintenance activities for each instrument, enabling upgrades to routine maintenance procedures, identification of opportunities for sequencing platform improvements, and more effective instrumentation purchasing decisions.

Primer walking of cloned DNA is a standard research tool. It has been used in the past to determine the sequence of individual clones of interest. With the expansion of DNA sequencing capacity the need to be able to walk larger numbers of clones has become necessary.

Our laboratory is a mid-sized genomics facility. In conjunction with the Advanced Biomedical Computing Center (ABCC) we have developed methods for automating the primer selection, DNA sequencing, contig assembly and sequence analysis for clones arrayed in microtiter format. This approach has allowed us to walk 475 clones (five microtiter plates) selected from a cDNA library.

The new Analyst™ GT is a multi-mode plate reader supporting all five leading technologies: fluorescence polarization (FP), fluorescence intensity, time-resolved fluorescence (TRF), absorbance and luminescence. It can be easily integrated into both workstation and robotic environments for HTS for all major non-radiometric applications. Analyst™ GT is based on second-generation SmartOptics™, a patented system that combines light sources, optical components and detectors together for fast high-precision measurements in 96-, 384-, and 1536-well microplates. Applications include kinase and photophase assays, receptor ligand binding assays, protease assays, cAMP, and cGMP, signal transduction, reporter gene assays and many others.

In this study, we not only optimized measurement parameters on Analyst™ GT for all five modalities but also compared instrument settings and performance with the first generation Analyst HT and Acquest.

Screening for ADME/Tox properties of compounds is a key step in the drug development process. Three applications defining such properties, namely Cytochrome P450 Inhibition, Serum Protein Binding and P-glycoprotein Interaction were developed and the performance of each was evaluated on Tecan's LabCD-ADMET System. The LabCD-ADMET System is an automated platform which integrates microfluidics, in the form of the centrifugally driven LabCD device; liquid handling and assay robotics, in the form of a modified GENESIS liquid handling workstation; an incubation, spinner, and detection system, the ULTRA LabCD; and platform/application-specific software. The assay approach and the data for each application are presented. Two applications, Cytochrome P450 inhibition and serum protein binding assays, have also been externally validated at leading pharmaceutical companies within Tecan's Early Access Program (EAP). The LabCD-ADMET System provides a miniaturized, integrated, and automated turnkey solution for various ADME/Tox applications.

It is estimated that about 18 million people worldwide suffer from dementia and it is projected to increase to about 35 million by the year 2025. All types of dementia occur due to an aberration in memory retention and development, caused by malfunctioning neurons. Experimental investigation of the dynamics of biological networks is a fundamental step towards understanding how the nervous system works. Activity-dependant modification of synaptic strength is widely recognized as cellular basis of learning, memory and developmental plasticity. Understanding memory formation and development, thus translates to changes in the electrical activity of the neurons. It is not possible to achieve this understanding at a cellular level by in vivo studies. To map the changes in the electrical activity it is essential to conduct in-vitro studies on individual neurons. Hence there is an enormous need to develop novel ways for assembly of highly controlled neuronal networks. To this end, we used a 5 × 5 multiple microelectrode array system to spatially arrange neurons, by combination of applied DC and AC fields We characterized electric field distribution inside our test platform by using two dimensional finite element modeling (FEM). As the first stage in the formation of a neural network dielectrophoretic AC fields were used to position the neurons over the electrodes. We used DC electric field to control axon growth direction within the network. Applied electric field direction is found to be an important parameter for axon growth. Electrical impulses were recorded from the individual neurons in the network during positioning and network formation.

Amethod has been developed for fabricating polymer microstructures based on electric field induced self assembly and pattern formation. A dielectric fluid placed in between two conductive plates experience a force in an applied electric field gradient across the plates, which can induce a diffusive surface instability and self construction of the fluid surface. This process is exploited for the fabrication of self assembled polymer microstructures as well as replicated patterns through the use of pre-patterned plates or electrodes. FEM simulation is used to decide the minimum wavelength and electric gradient distribution of polymer structures. A variety of structures in the micron and nanometer scales including bio-fluidic MEMS, polymer optoelectronic devices can be fabricated using this method.

We have analyzed the detection of microcantilevers utilized in biosensing chips. First, the primary deflection due to the chemical reaction between the analyte molecules and the receptor coating, which produces surface stresses on the receptor side is analyzed. Oscillating flow conditions, which are the main source of turbulence in cantilever based biosensing chips, are found to produce substantial deflections in the microcantilever at relatively large frequency of turbulence. Then mechanical design and optimization of piezoresistive cantilevers for biosensing applications is studied. Models are described for predicting the static behavior of cantilevers with elastic and piezoresistive layers. Chemo-mechanical binding forces have been analyzed to understand issues of saturation over the cantilever surface.

Furthermore, the introduction of stress concentration regions during cantilever fabrication has been discussed which greatly enhances the detection sensitivity through increased surface stress, and novel microcantilever assemblies are presented for the first time that can increase the deflection due to chemical reaction. Finally an experiment was made to demonstrate the shift of resonant frequency of cantilever used as biosensor. The relation between resonant frequency shift and the surface stress was analyzed.