Journal of Laboratory Automation

The use of cell colony formation assays for research and clinical applications to assess the functional integrity of cells after in vitro manipulations is extensive. Key areas include hematopoietic stem cell research, cell transformation studies, and predicting the response of tumors to chemotherapeutic agents. Traditionally, enumeration of colonies has involved laborious and subjective counting by hand using a microscope. Here, laser scanning microplate cytometry has been used to provide an automated high-content readout of the effects of cytostatic agents on colony formation. This approach determines colony number through the application of a volume algorithm. Such an approach permits the differentiation of cytostatic effects where the number of colonies and size remains constant, and cytotoxic effects where the size and number may be reduced. Application of microplate cytometry thus offers significant benefits over alternative analytical methods in the search for novel chemotherapeutic agents.

There is a growing need to precisely quantify the selectivity of large compound sets in high throughput screening, directing investment in lead optimization towards compounds with a high chance of success. High-content, high-density screening technologies such as multiparametric ultra-HTS provide a basis for highly precise screening with unprecedented scope for delineating process artifacts from reliable signals. However, the full potential of these technologies can only be realized with suitable experimental design and sophisticated data analysis tools.

We present two advanced analysis workflows demonstrating how multiparametric readouts from a high throughput primary screen can improve decision quality in the hit identification process. The first involves discrete thresholding and the application of multiple selection criteria. The second uses machine learning algorithms and allows an unbiased consideration of all measured parameters.

Theoretical studies focusing on the nature of landscapes that correlate molecular sequences to molecular function have mainly been carried out in silico due to the vast amounts of data that are needed. Automated in vitro selection is capable of producing significant amounts of data in a short time, making theoretical modeling with real experimental data attainable. A Biomek 2000 Laboratory Automation Workstation has been outfitted to carry out multiple in vitro nucleic acid selections in parallel, yielding substantial amounts of data for theoretical studies. A random sequence population of nucleic acids is initially generated by a combination of chemical synthesis and enzymatic amplification. On the workstation, this population is parsed for its ability to bind a protein, lysozyme. After each round of selection, the selected nucleic acid binding species (also known as aptamers) are amplified by a combination of reverse transcription polymerase chain reaction (PCR) and in vitro transcription. All eight pools that have undergone selection have yielded different sequences.

In this paper we present the novel adjacent impedance probing (AIP) technique for DNA hybridization detection. In our design, the DNA hybridization site was employed only for the biorecognition event (this site does not necessarily need an underlying conductor surface). A bare adjacent electrode was used for detection of an impedance change. An enzymatic reporter produced the deposition of an insulator on the adjacent electrode. The adjacent impedance probing (AIP) technique is employed to alleviate the low-signal or high-noise problems caused by ssDNA capture probes adsorbed (nonspecifically through non-Au-S binding) on the electrode surface. Data showed that hybridization resulted in a twofold increase in impedance.

PatchXpress, an automated 16-channel parallel patch clamp system, was used to determine inhibition of human ether-a-go-go related gene (hERG) potassium channels by known blockers. A monoclonal cell line stably expressing hERG potassium channels was generated in CHO-KI cells. Results were compared to conventional patch clamp experiments using similar voltage protocols and solutions. Success rates were evaluated for cell recordings under a variety of conditions, including Accumax versus trypsin treatment to harvest cells, single versus double compound additions, and polystyrene versus glass-coated compound plates. We found that the average success rates rose from 27% with trypsin treatment to 38% with Accumax treatment, which improved to 55–65% following long-term culturing using only Accumax to harvest cells. Two drug additions (spaced 1 min apart with suction off) were also found to produce data that more closely matched conventional experiments. Finally, polystyrene versus glass-coated compound plates were evaluated, and we found that for some compounds (but not all), preparation of compound samples in glass-coated plates resulted in inhibition that more closely matched data obtained by conventional experiments. Therefore, we have established an assay to evaluate the ability of compounds to inhibit hERG potassium channels, which closely matches data produced using conventional methods but with much greater throughput.

Forensic labs globally face the same problem—a growing need to process a greater number and wider variety of samples for DNA analysis. The same forensic lab can be tasked all at once with processing mixed casework samples from crime scenes, convicted offender samples for database entry, and tissue from tsunami victims for identification. Besides flexibility in the robotic system chosen for forensic automation, there is a need, for each sample type, to develop new methodology that is not only faster but also more reliable than past procedures. FTA is a chemical treatment of paper, unique to whatman Bioscience, and is used for the stabilization and storage of biological samples. Here, we describe optimization of the Whatman FTA Purification Kit protocol for use with the AmpFlSTR Identifiler PCR Amplification Kit. The conditions giving the best quality Identifiler results were used to automate a simple rapid method that processed forensic samples applied to FTA-treated paper punches in a 96-well plate in ∼30 min. We describe an automated DNA purification procedure using 1.25 mm bloodstained FTA punches that is easily scalable and can be used with both convicted offender and reference casework bloodstained and buccal-swiped FTA samples. It works equally well with the Profiler Plus PCR Amplification Kit. Fully automated drying and polymerase chain reaction (PCR) amplification setup of short tandem repeat (STR) loci can be added to expand the walk-away process.

The optimization of synthesis conditions plays an important role in chemistry and life sciences. A wide variety of reaction parameters should be explored to minimize the use of educts and maximize the yields of desired products in high purity. A fully automated system for combinatorial synthesis has been developed and used for the optimization of synthesis conditions of a multicomponent single-pot reaction. In order to explore optimal reaction conditions for the synthesis of pure endo isomer of 3-(2-bromo-benzoylamino)-cyclohex-4-ene1,2-dicarboxylic acid diethyl ester, an intermediate in the synthesis route of lycorizidine analogues, several hundred experiments were carried out with varying reaction temperature, times, concentrations, and ratios of educts. In this case study, financial aspects also were considered in order to minimize synthesis costs.

The system was integrated using a Zymate XP robot as a system integrator. In addition to a GC/MS and an HPLC system, a multicomponent solid dispenser workbench and a liquid handling system are located within this fully automated system.

Simple sequence repeats (SSR) or simple sequence length polymorphisms (SSLP) are PCR-based molecular markers that have been widely used in genomic mapping and marker-assisted selection. SSR markers have made it possible to establish a high-density genetic map and evaluate genes of interest via tight association between markers and phenotypes. Large-scale markerassisted breeding studies in plants have created the need for a high-throughput system for genomic DNA preparation and analysis from thousands of samples in a segregating population. However, the standard methods for purifying DNA from plant tissues can be laborious, time-intensive, and not readily amenable to automation. Thus, an automated system for the rapid extraction and subsequent amplification and analysis of plant genomic DNA has been developed to facilitate high-throughput genome mapping and marker-assisted breeding studies. This system utilizes Sigma's Extract-N-Amp Plant PCR kit, a novel system for the rapid extraction and subsequent amplification of genomic DNA from plant tissues, and the maize SSR primer set. This extraction system eliminates time-consuming steps such as organic extractions and mechanical disruption, releasing sufficient genomic DNA from plant tissues for direct use in SSR marker analysis.

The Electron Microscopy Proteomic Organellar Preparation (EMPOP) robot is a tool for high-throughput preparation of subcellular fraction samples for electron microscopic identification. It provides a means of validating subcellular sample purity and confirming protein localization needed for organellar proteomics.

The device handles all chemical and mechanical manipulations required to prepare organelles for electron microscopic examination. It has a modular, integrated design that supports automated filtration, chemical processing, delivery, and embedding of up to 96 subcellular fraction samples in parallel. Subcellular fraction specimens are extremely fragile. Consequently, the system was designed as a single unit to minimize mechanical stress on the samples by integrating a core mechanism, composed of four modular plates, and five support subsystems: (1) a cooling platform, (2) an automated fluid handling subsystem, (3) an electromagnetic arm, (4) a plate transfer platform, and (5) a 5-axis motion control system (X, Y, Z, θ, ø).

System control is fully automated to provide standardized, reproducible subcellular fraction sample processing while maintaining flexibility for adjustment and recall of instrumentation and process operational parameters. To achieve this, the control software was built on two coordinated levels: (1) a user interface for system testing, calibration, setup, and process monitoring and (2) low-level real-time control routines.

The EMPOP robot provides, for the first time, massive, parallel electron microscopic screening and quantitative analysis of subcellular and protein targets necessary for high-throughput proteomics.

In the quest for novel pharmaceuticals to treat important diseases, biologists often use in vitro assays to assess apparent activity. Thus, when testing novel synthetic compounds in search of a new class of drugs, it is important to know the solubility of the compound under the assay conditions used so that the activity data may be interpreted correctly. As the screening throughput increases, so do the demands on solubility determinations and the reliance on this data to aid in the selection of the more promising compounds for further studies. Since many compounds synthesized in drug discovery compounds contain UV chromophores, solubility determinations using UV plate readers are widely applicable to common druglike compounds. A more analytically rigorous approach calculates the solubility using a calibration curve rather than a ratio to a reference concentration. Rather than use the λ_max of the analyte for quantification, an automated method is used for intelligent selection of the wavelength most appropriate for quantification. Using a BioMek FX and Peak Seeker, our visual basic application written in-house, 128 compounds may be assayed at a single pH within 5 h using MultiScreen Solubility plates.

Cellular dielectric spectroscopy (CDS) provides realtime, label-free, universal measurements, enabling comprehensive pharmacological evaluation of cell surface receptors in living cells. The sensitivity of the measurement allows monitoring of ligand-mediated activation of endogenous receptors, therefore generating physiologically relevant data. Activation of receptors results in CDS response profiles that are characteristic of main subsets of G-protein coupled receptors (GPCRs) within a cell line. This allows cluster analysis of response profiles that may be used in several important applications, which include identification of the G-protein coupling of orphan GPCRs and the cataloging of active endogenous receptors in cells. In this study, CDS technology is used in the pharmacological evaluation of multiple receptors in many cell types, including primary cells. Specifically, data is presented demonstrating hit confirmation, receptor selectivity analysis, ligand potency, and Schild analysis of receptor-selective antagonists. CDS results compare favorably to other cell-based assays, and the robustness and reproducibility of CDS assays are reflected by low assay coefficient of variation (CVs) and reliable Z'-scores of the data. Because CDS requires no stable or transiently transfected cells or special reagents, assay development and data acquisition is simple and fast. The ease of use, universality, and label-free nature of the CDS-based platform make it well suited to secondary screening applications in drug discovery.

The red blood cells (RBCs) from patients with such diseases as pulmonary hypertension (PH) and diabetes (Type I and II) have a unique physical property, namely, the deformability of their RBCs are different from those of healthy patients. For example, patients with PH and diabetes have RBCs whose deformability is less than that of healthy patients. Unfortunately for these patients, there have been very few reports relating these somewhat abnormal RBC deformabilities to a cause/effect of the aforementioned diseases. Our group believes there is a link between the physical properties of these RBCs, the chemical properties they induce, and the resultant physiological consequences of the induced chemical properties. However, characterization or verification of the physical, chemical, and physiological processes is difficult because current analytical tools do not enable all three processes to be monitored simultaneously. Here, the ability of microchip technology to monitor physical, chemical, and physiological events that may occur in each of the diseases will be discussed. These events, which are physically stimulated on-chip, are monitored both optically (chemiluminescence) and electrochemically (amperometry), and when taken collectively, become excellent in vitro models of important in vivo processes.